The Epicardium in Cardiac Repair From the Stem Cell View

Cardiovascular disease caused by ischemic injury remains the leading cause of death worldwide.¹ Millions of cardiomyocytes are lost to myocardial infarction (MI), which rapidly triggers an innate immune response to clear dead or dying cells and activate collagen-producing myofibroblasts to remodel the extracellular environment.² Cardiac muscle damage is irreversible as adult mammalian cardiomyocytes do not re-enter the cell cycle to replace ischemic tissue.³ Over the last decade, several strategies have been used to promote cardiac repair including stimulation of cardiomyocyte cell cycle re-entry, delivery of stem cells, or stem cell-derived cardiomyocytes, modulation of tissue repair using cell-free therapeutics, and applying viral vectors and bioengineered patches to deliver proregenerative factors.^3–5 However, it is becoming apparent that cardiac repair will require active contributions from numerous cardiac cell types.

The epicardium is an evolutionarily conserved layer of mesothelium covering the outermost cell layer of the vertebrate heart. During fetal development, the epicardium serves as a progenitor source, contributing multipotent cells that give rise to cardiac mesenchyme. However, the role of the epicardium is not limited to de novo formation of nonmyocyte lineages. This mesothelial layer also fosters a paracrine milieu critical for myocardial growth and coronary vessel patterning. As the heart matures, the epicardium undergoes a period of dormancy, functioning as a simple barrier between the myocardium and the pericardial cavity. However, recent studies in zebrafish and mice have elegantly shown that cardiac injury reanimates the epicardium and its derivatives to modulate tissue repair.⁶ This review will summarize the dynamic roles of the epicardial lineage during heart development with a focus on developmental programs that may be harnessed to facilitate cardiac repair.

The Epicardium: From Origin to a Progenitor Source

Formation of a functioning heart requires the assembly and integration of multiple progenitor cell populations that arise from embryonic structures, such as the cardiogenic mesoderm, the cardiac neural crest, and the proepicardium.⁷ The proepicardium is an evolutionarily conserved and transient cluster of progenitor cells located at the venous pole of the fetal heart, which has been described in many vertebrate species ranging from lampreys to humans.^8–13 Proepicardium-derived cells exhibit a migratory event at embryonic day (E)9.5 in mice and Hamburger-Hamilton stage 17-18 in chick, translocating from the proepicardium and towards the atrioventricular canal (in chick) of the nascent heart. Here, proepicardium-derived cells form a single-cell layer of mesothelium, called the epicardium, that lines the outermost layer of the heart.^11,12,14 Initially, the epicardium exhibits epithelial-like properties; however, a subset of epicardial cells delaminate from the epicardial sheet and invade the myocardium generating a majority of vascular smooth muscle cells (SMCs) and cardiac fibroblasts in the heart.¹⁵ Thus, the epicardium is critical for providing support cells that contribute to myocardial integrity.

Epicardial Epithelial-to-Mesenchymal Transition

Epicardium-derived progenitor cells (EPDCs) arise from the epicardial layer through an epithelial-to-mesenchymal transition (EMT) event that initiates after Hamburger-Hamilton stage 18 in chick and approximately E12.5 in mice.^16,17 In human, expression of EMT markers have been observed in the expanded epicardium surrounding the ventricles at fetal stage 3 (equivalent to E17.5-18.5 in mouse).⁸ EMT is an evolutionarily conserved process by which epithelial cells lose their apical-basal polarity and cell-cell adhesions and acquires migratory and invasive characteristics akin to multipotent mesenchymal stem cells.¹⁸ Progenitor cells that emerge from EMT often give rise to new cell lineages during organogenesis. Early studies in chick embryos used dyes or retroviral vectors to label epicardial cells, which demonstrated the incorporation of EPDCs in cardiac mesenchyme and coronary vasculature.^19–21 Epicardial EMT and epicardial-derived cell fates (Figure 1) were subsequently described in mice using genetic fate-mapping approaches that indelibly label all progeny of Cre recombinase-expressing cells with β-galactosidase (LacZ) or a fluorescent lineage reporter. This technology uses specific regulatory sequences to drive the expression of Cre in the epicardium of transgenic mice. Importantly, while it appears that no single gene exclusively marks the epicardium, several gene regulatory sequences have been used to trace epicardial cells and mesothelium-derived cells found in other tissues, including Scx, Sema3d, Gata5, Tbx18, Tcf21, and Wt1. The use of tamoxifen-inducible Cre alleles (Cre recombinase fused to a mutant estrogen ligand-binding domain or CreERT2) has also provided temporal control to study the timing of epicardial EMT and to fate map descendants of the epicardium.²²Tables 1 and 2 summarize the current genetic animal models available and the cells and tissues labeled by each marker.

Table 1. Epicardial Lineage-Tracing Strategies

Transgenic Model	Organism	Description	Cardiac Cell Fates Mapped
G2-Gata4Cre; G2-Gata4LacZ 23	Mouse	Constitutive, transgenic line expressing the conserved CR2 enhancer of Gata4	EP, EC, SMC23
cGata5Cre 24	Mouse	Constitutive, transgenic line expressing chicken Gata5 promoter fused to Cre	EP, SMC, CF24
Wt1CreERT2 15	Mouse	Inducible, CreERT2 knockin into the Wt1 locus	EP, SMC, EC, CM, CF, VIC and PC (TAM E9.5—E14.5)15,25–27 AC (Re),28,29 Mes (Re)30
Wt1Cre 31	Mouse	Constitutive, BAC transgenic—IRES/EGFP-Cre inserted downstream of the Wt1 translation stop site	EP, CF, VIC and CM,31 CFs (Re)32,33
Wt1GFPCre 15	Mouse	Constitutive, GFP-Cre fusion knockin into the Wt1 locus	EP, CM15
Tbx18CreERT2 34	Mouse	Inducible, CreERT2 knockin into the Tbx18 locus	PC and SMC34
Tbx18Cre 35	Mouse	Constitutive, Cre knockin into the endogenous Tbx18 locus	EP, SMC, PC, CF, and CM,35,36 CFs (Re)37
Tcf21iCre 38	Mouse	Inducible, MerCreMer knockin into the Tcf21 locus	EP, CF, SMC (TAM E10.5, E14.5)39 and CF (Re)40
Tcf21Lacz 41,42	Mouse	Constitutive knockin into the Tcf21 locus	EP and CF39
tcf21:CreER43	Zebrafish	Inducible, BAC transgenic—CreERT2 knockin into the tcf21 locus	EP and SMC; EP and PV (Re)43
tcf21:dsRed43	Zebrafish	Constitutive	EP, PV, and SMC; Mes and PV (Re)43
ScxGFPCre 44,45	Mouse	Constitutive	EP, EC, SMC, and CM,44 VIC45
Sema3DGFPCre 44	Mouse	Constitutive	EP, EC, SMC, and CM44

Table 2. Labeling of Cre Lines in Noncardiac Tissue

Gene	Noncardiac Tissue Labeling
WT1	Adrenal gland and kidney,46 gut,58 liver,59 lung,60 diaphragm,61 and adipose tissue62
TBX18	Skeletal muscle, brain, retina, lymph nodes, skin, sinoatrial node, adipose tissue, and bone marrow34
GATA5	Septum transversum, liver, and kidney24
TCF21	Lung, kidney, gonads, spleen, gut, adrenal gland, and skeletal muscle38,41,48
SCX	Tendons and ligaments63
SEMA3D	Pharyngeal arch endoderm44

• Download figure
• Download PowerPoint

Figure 1. Epicardium during cardiac development. EPDCs (Epicardium-derived progenitor cells) emerge from the fetal epicardium through the process of epithelial-to-mesenchymal transition (EMT) and contribute to various cardiac lineages, such as cardiac fibroblasts (CFs), smooth muscle cells (SMCs), and pericytes (PCs). The ability of EPDCs to differentiate into endothelial cells (ECs) in vivo is limited, and cardiomyocytes (CMs) are not generally thought to derive from EPDCs. In addition to cellular contributions, the epicardium participates in reciprocal paracrine signaling (dashed arrows) to stimulate CM proliferation, macrophage (MC) recruitment and coronary vessel growth and maturation. Epi indicates epicardium; myo, myocardium; and subepi, subepicardium. Illustration credit: Ben Smith.

Molecular Regulation of Epicardial EMT

Transcription factors (TFs) mediate transdifferentiation of epithelial cells into a mesenchymal phenotype through the repression of genes encoding epithelial adhesion molecules (E-cadherin, claudins, and occludens) and activation of mesenchymal genes (N-cadherin, collagens, and FN [fibronectin]) necessary for ECM (extracellular matrix) production and migration.¹⁸ Here, we will highlight known transcriptional regulators, as well as upstream growth factor responsive pathways, implicated in epicardial EMT (Figure 2).

• Download figure
• Download PowerPoint

Figure 2. Molecular control of epicardial epithelial-to-mesenchymal transition (EMT). Intrinsic and extrinsic molecular programs regulate epicardial EMT in mice, which include the regulation of transcription factors and molecular signaling. Cytokine-mediated signaling between the epicardium and cardiomyocytes is reported to stimulate heart growth, and epicardium-derived signals support the growing coronary vasculature. CXCL, C-X-C motif chemokine receptor ligand; CXCR, CXC chemokines and receptor; FGF, fibroblast growth factor; IGF, insulin growth factor; MRTF, myocardin-related transcription factor; PDGFR, platelet-derived growth factor receptor; TGF, transforming growth factor; and YAP, yes associated protein 1.

Wt (Wilms tumor)-1 is a zinc finger TF that is highly expressed in the epicardium during heart development and downregulated concomitant with epicardial EMT.^46,64 Global or conditional deletion of Wt1 in mice results in embryonic lethality during mid to late gestation and hearts display defective epicardial EMT and EPDC invasion, which leads to myocardial hypoplasia and abnormal coronary vascular plexus coverage.^65,66 These defects may be attributed to altered transcription as WT1 regulates numerous targets during EMT,^52,66–68 including activation of Snai1, which represses the expression of epithelial Cdh1 (E-cadherin).⁶⁵ Wt1 has also been shown to act upstream of canonical Wnt/β-catenin and retinoic acid (RA) signaling, both critical for epicardial biology and EMT.¹⁷ Consequently, loss of β-catenin in the epicardium disrupts adherens junctions and cell polarity, ultimately impairing EPDC migration and coronary artery formation.^17,69

Tcf21 (Pod1/epicardin/capsulin) is a class II basic helix-loop-helix TF highly expressed in the proepicardium and epicardium.⁴⁷ Tcf21-depleted Xenopus embryos fail to form a cohesive sheet of mesothelial cells lining the heart, suggesting that Tcf21 is critical for proepicardial cell migration and proper formation of the epicardium.¹³ In mice, Tcf21 regulates epicardial EMT as evidenced by downregulation of Snai1, Sox9, and Zeb1 in Tcf21 null epicardial explants.³⁹ The exact mechanisms linking Tcf21-dependent transcription to epicardial homeostasis and EMT remain unclear, although interactions with transcriptional co-repressors and direct binding to E-box consensus sequences (CAnnTG) may contribute to these processes.^13,70

Regulation of EMT and motility gene programs by Hippo signaling and MRTFs (myocardin-related transcription factors)/SRF (serum-response factor) are required for the invasion of EPDCs into the myocardium. Yap (Yap1) and Taz (Wwtr1) are evolutionary conserved co-factors negatively targeted by the Hippo pathway to restrain organ size.⁷¹ In addition to regulating contact inhibition, cell proliferation and stem cell self-renewal, Yap and Taz also mediate EMT in various contexts.⁷¹ Conditional deletion of Yap and Taz using the Wt1 ^CreERT2 allele diminishes the expression of multiple EMT associated TFs, including Snai1, Snai2, Twist1, Wt1, and Tbx18.⁷² Interestingly, the Hippo pathway must be tightly controlled in the epicardium as Yap/Taz-deficient (active Hippo) or Lats1/2-deficient (inactive Hippo) murine EDPCs exhibit defects in cell migration and fibroblast differentiation.^72,73

SRF is a TF that is essential for the formation of the proepicardium and epicardial EMT.^74,75 SRF binds to a DNA motif called a CArG box [CC(A/T)₆GG], activating gene programs related to actin cytoskeletal rearrangements, cell adhesion and migration, and vascular smooth muscle cell commitment.^74–77 Nuclear SRF in epicardial cells corresponds with increased levels of smooth muscle genes, whereas the expression of dominant-negative SRF inhibits smooth muscle formation.⁷⁵ SRF transcriptional activity is greatly enhanced by interactions with the MRTF family of signal responsive co-factors.⁷⁸ MRTFs are tethered in the cytoplasm via interactions with monomeric actin, which conceals a nuclear localization signal.⁷⁹ Mechanical stretch or pathways that activate Rho-kinase and actin polymerization lead to nuclear accumulation of MRTFs and activation of SRF-dependent gene programs.⁷⁹ MRTF deletion using the Wt1 ^CreERT2 allele results in decreased migration of EPDCs during developmental EMT coupled with delayed maturation of the coronary plexus.²⁵ These findings highlight the importance of signal responsive transcriptional regulation in epicardial EMT and EPDC differentiation. Generally, EPDCs display heterogeneous expression of epicardial TFs in both the mouse and chick epicardium⁸⁰; thus, the diverse epicardium-derived lineages described in this review may arise, in part, because of intrinsic differences in EPDCs that modulate the response to paracrine cues.

Growth Factor-Dependent Regulation of Epicardial EMT

TGF (transforming growth factor)-β is among the most well-studied upstream regulators of EMT and ECM production.⁸¹ TGF-β1-3 are all expressed in the mouse epicardium by E12.5⁸² and targeted deletion of type I TGF-β receptor Alk5 using the chicken Gata5 enhancer (cGata5)-Cre perturbs epicardial EMT, leading to myocardial hypoplasia, and attrition of SMCs and microvascular structures.⁸³ Likewise, global deletion of type III TGF-β receptor (Tgfbr3 ^−/− mouse model) abrogates the coronary plexus, which is attributed to dysregulated inflammatory signaling in the epicardium and defects in bone morphogenetic protein 2-mediated EPDC motility.^84,85 Mouse and human epicardial explants stimulated with TGF-β1 and TGF-β2 lose epithelial markers (tight junction protein Zonula occludens-1 and cytokeratin) and upregulate SMC markers (α-smooth muscle actin, transgelin or SM22, calponin, and caldesmon) indicative of EMT.^86,87 TGF-β2 stimulation also amplifies epicardial motility by indirectly stimulating expression of hyaluronan synthase 2 (Has2) and the production of the ECM component hyaluronan.⁸⁸

PDGF (Platelet-derived growth factor) signaling is ubiquitous during mouse cardiogenesis and regulates critical processes, such as cardiac neural crest cell recruitment to the outflow tract, coronary vessel maturation, and myocardium development.^89–91 PDGFR (PDGF receptor)-α and PDGFR-β are receptor tyrosine kinases that play critical roles in regulating epicardial EMT and EPDC specification.⁹² Epicardial cells null for Pdgfra/b are unresponsive to pro-EMT stimuli (TGF-β, fibroblast growth factor [FGF]-2, and PDGF-BB) and express low levels of EMT TFs (Snai1 and Sox9) and mesenchymal genes.⁹² Defects in EMT can be rescued by forced overexpression Sox9 in PDGFR mutant cells, suggesting that Sox9 is downstream of PDGFR-α/β signaling.⁹² Conditional or global deletion of each receptor separately or together, using cGata5-Cre or WT1 ^CreERT2 alleles, limits EPDC motility by disrupting phosphoinositide 3-kinase-mediated regulation of actin polymerization.^92–94

Epicardium-Derived Interstitial Lineages

Various cardiac interstitial cell lineages, including fibroblasts, vascular mural cells, and to a limited extent, endothelial cells (ECs), arise from the epicardium (Figure 1).

Cardiac Fibroblasts

Lineage tracing and reporter mouse lines, such as Tcf21 ^MerCreMer/+ ;Rosa26 ^EGFP ,³⁸ Collagen1a1 (Col1a1)-GFP,^32,95 and PDGFRα ^GFP/+ ,⁹⁶ facilitate the identification of resident cardiac fibroblasts originating from EPDCs,⁹⁷ which are especially populous in regions near the sinoatrial node, atrioventricular cushions and valves, and annulus fibrosis.^26,31,98,99 Notably, Tcf21 controls the specification of EPDCs to SMC or fibroblast lineages during murine embryonic development.¹⁰⁰ Tcf21 null hearts have fewer Col1a1 (collagen1a1)-GFP⁺ fibroblasts, while the number of SMCs remains unaltered.³⁹ In contrast, activation of Tcf21 through exogenous RA to embryonic chick hearts reduces the number of SMCs.⁸⁰ In addition to Tcf21, upstream cues such as PDGF signaling can influence cardiac fibroblast cell fate. PDGFR isoforms display overlapping expression patterns in the early stages of epicardial formation but become distinct by E13.5 in murine models.⁹² Pdgfra loss-of-function impairs EPDC differentiation into cardiac fibroblasts while conditional deletion of Pdgfrb impacts the vascular SMC lineage.⁹² These findings suggest that PDGFR-α and PDGFR-β regulate distinct epicardium-derived developmental trajectories.

Vascular Mural Cells

Vascular SMCs lining the coronary arteries were first demonstrated to arise from proepicardium-derived cells in avian models.¹⁹ These findings were later substantiated in mice using Cre-dependent tracing of cells originating from Tcf21,⁹² Wt1,¹⁵ and Tbx18 ³⁵-expressing progenitors. Tbx (T-box protein)-18 belongs to a large family of T-box TFs that regulate a variety of developmental processes in vertebrates and invertebrates and is enriched in the proepicardium, epicardium, subepicardial mesenchyme, septum transversum, and a subset of the myocardium.^49,50,101 Tbx18 is dispensable for epicardial development and EMT; however, loss of Tbx18 results coronary vessel maturation defects.^49,102 Forced expression of Tbx18 prevents SRF-dependent activation of smooth muscle reporters.¹⁰² Conversely, conditional misexpression of Tbx18VP16 (fusion of the Herpes simplex virus-encoded protein VP16 to the C-terminus of Tbx18) results in hypoplastic hearts and ectopic smooth muscle differentiation mediated by Notch3 and TGF-β signaling.^103,104 Mature SMCs (smooth muscle myosin heavy chain, also known as MYH11⁺) are reported to arise via the differentiation of Tbx18 lineage-derived pericytes,³⁶ which are stellate-like cells that encapsulate the microvasculature and preserve vascular integrity. Epicardium-derived cardiac pericytes, as defined by expression of PDGFR-β and chondroitin sulfate proteoglycan 4 (also known neuron-glial antigen 2), are required for the stability and growth of the embryonic coronary plexus.^25,105 Indeed, EMT deficiency associated with genetic deletion of MRTFs in the epicardium depletes the heart of cardiac pericytes, leading to reduced vascular integrity and subepicardial hemorrhage.²⁵

Vascular ECs

Coronary ECs are predominately derived from nonepicardial sources.¹⁰⁶ Lineage-tracing studies in mice have excluded epicardial-derived ECs from Tbx18, c-Gata5, or Tcf21 lineage-derived EPDCs.^24,35,39 However, Wt1-derived epicardial cells have been shown to give rise to ECs in the developing chick embryo²¹ as well as EPDCs fate mapped from the Wt1 ^CreERT2 lineage after administration of tamoxifen at E10.5 or early postnatal time points in murine models.^15,27 However, the contribution of the epicardium towards the EC fate has been challenged by reports of endogenous Wt1 expression in mature endothelium beginning at E12.5 and persisting throughout postnatal mouse development.⁵⁷ Scleraxis (Scx) and Sema3D positive epicardial cells form rare populations of ECs validated by in vivo fate mapping and in vitro cultures.⁴⁴ While the Sema3D ^GFPCre , Scx ^Cre , Wt1 ^CreERT2 , and G2-Gata4 ^Cre epicardial Cre lines^23,44 may sporadically label the endothelium, the majority of cardiac ECs are derived from the sinus venosus and endocardium.¹⁰⁵

The Epicardium-Derived Secretome

The previous sections highlight the complexity of epicardial EMT and epicardium-derived cell fate determination, which are controlled by both cell autonomous and cell nonautonomous mechanisms. The following sections will discuss some of the intercellular signaling cascades underlying paracrine regulation of cardiomyocyte and vascular development and homeostasis (Figure 2).

Epicardium-Cardiomyocyte Crosstalk

In addition to directly differentiating into various cell lineages, the epicardium also supports cardiomyocyte proliferation and embryonic heart growth by providing a cocktail of secreted mitogens. FGF-1, -2, -4, -6, -9, and -16 are all expressed by the fetal epicardium.^107,108 In particular, both endocardial and epicardial FGF-9 signals regulate cardiomyocyte proliferation via activation of fibroblast growth factor receptor (FGFR)1c and FGFR2c.¹⁰⁷ In contrast, FGF-10 secreted from the myocardium promotes EPDC invasion and EPDC-to-fibroblast conversion by signaling through FGFR2b expressed on epicardial cells.¹⁰⁹ The mitogenic activity of cardiomyocytes can be stimulated by the secretion of IGF (insulin growth factor)-2 from epicardial cells in vitro, a phenomenon that is recapitulated through the pro-proliferative signals emanating from the developing epicardium in vivo.^110,111 In fact, global deletion of Igf2, or conditional deletion of its receptor Igfr1 in Nkx2.5 expressing cardiomyocytes, results in severe cardiac defects in the form of ventricular noncompaction.^111,112

RA signaling is critical for proper cardiac morphogenesis^113,114 and can induce the expression of epicardial-restricted TFs, such as Wt1 and Tcf21.⁸⁰ RA regulates transcription by binding to nuclear RA receptors which form heterodimers with RXR (retinoid X receptor).¹¹⁵ RXRα ^−/− mice display normal cardiac mouse development up to E11.5; however, thereafter fail to undergo proper myocardial compaction and become severely hypoplastic.^113,114 RA signaling stimulates epicardial secretion of trophic signals such as WNT-9A, FGF-2, FGF-9, and IGF-2 to promote myocardial growth and proper coronary vessel maturation.^{24,107,110,111} RA is synthesized by the oxidation of retinaldehyde by Raldh (retinaldehyde dehydrogenases)-1, -2, and -3,¹¹⁵ which are highly expressed in the fetal mouse epicardium at E12.5.⁵³ Global deletion of Raldh2 results in embryonic lethality around E9.5 with a single dilated cardiac chamber.¹¹⁶ Remarkably, this phenotype can be partially rescued by maternal supplementation of exogenous RA.¹¹⁶

Epicardium-EC Crosstalk

The epicardium is also an important source of chemokines that regulate coronary vessel patterning. Prevention of proepicardial outgrowth and genetic ablation of epicardial cells with diphtheria toxin results in disorganized coronary vasculature and reduced recruitment of subepicardial cardiac macrophages.^117,118 Wt1 knockout cells also express lower levels of vascular growth factors such as angiopoietin-1 and VEGF-A (vascular endothelial growth factor A),⁶⁶ and Wt1 is needed to suppress the production of proinflammatory cytokines chemokine ligand 5 and CXCL (C-X-C motif chemokine ligand) 10 in epicardial cells.⁶⁷ Thus, the epicardium serves as an essential secretory hub to maintain vascular and inflammatory cell stability in the fetal heart. ECs also communicate with the developing epicardium by facilitating the induction of EMT and differentiation towards mural cell lineages via activation of PDGFR-β.¹¹⁹ Pericyte recruitment to the microvasculature is coordinated in part by the expression of the Notch ligand Jagged-1 in ECs activating Notch3 receptors on epicardial-derived pericytes.³⁶ CXCRs (CXC chemokines and receptors), such as the CXCL12-CXCR4 axis, have been shown to play an essential role in coronary artery maturation.¹²⁰ Cxcr4 in zebrafish is expressed in angiogenic ECs and mature coronary arteries lined by mural cells, whereas cxcr4 deletion results in disorganized angiogenic sprouts.¹²⁰ In contrast, Cxcl12 is expressed in the epicardium and epicardial-derived mural cells in both zebrafish and mouse models.^120,121 Although localized expression of CXCL12 is sufficient to increase capillary density through the recruitment of CXCR4⁺ ECs, global myocardial overexpression disrupts vascular formation.¹²⁰ These findings indicate that discrete gradients of chemotactic cues are required during the intricate process of coronary vessel formation.

Overall, these data support a critical cell nonautonomous role for the epicardium in the fetal heart. Thus, the role of epicardial EMT appears to be 2-fold: (1) contribution of multipotent progenitors and (2) secretion of growth factors necessary for embryonic heart formation (Figures 1 and 2). Harnessing fetal epicardium programs that build the developing heart may have therapeutic implications for the stimulation of scarless cardiac repair through regenerative medicine, which is discussed below.

Overview of Cardiac Repair and Regeneration

Advances in emergency medicine and cardiology have led to an abundance of patients surviving an MI. However, these successes have revealed deficiencies in supporting cardiac repair after an initial injury, which results in a growing population living with chronic heart disease. Adult mammalian cardiomyocytes are postmitotic with a limited capacity for self-renewal following acute insult or over periods of chronic disease.¹²² Therefore, progressive loss of cardiomyocytes via apoptotic or necrotic cell death leads to the replacement of damaged contractile tissue with a fibrotic scar, irreversible cardiac remodeling, and organ failure.² Recent studies have highlighted that cardiomyocyte renewal can be enhanced through molecular signaling pathways converging on the induction of critical cell cycle regulators (eg, cyclin A2), growth factors (neuregulin1, IGF-1/2), and intrinsic signaling pathways (eg, Hippo).³ Therefore, understanding endogenous cardiac repair mechanisms is an emerging topic of intense investigation that may lead to new regenerative medicine strategies.

Reactivation of the Adult Epicardium

Fetal epicardial gene activation after myocardial injury is thought to represent a conserved cardiac repair mechanism.¹²³ Wt1, Tbx18, Raldh1, and Raldh2 transcripts are all transiently upregulated between 1 and 5 days after MI in mice, particularly surrounding the valves and atria, and wane in expression by 2 to 4 weeks after injury.^30,124 Fetal epicardial gene induction correlates with increased proliferation and pronounced epicardial thickening, as documented in both adult mice and canine models.^30,124 Unlike the fetal epicardium, which undergoes EMT and generates cardiac fibroblasts and vascular mural cells, lineage-tracing experiments reveal minimal contribution of the epicardium to new adult fibroblasts or to collateral formation (Figure 3). In contrast, the adult epicardium is thought to serve as a source of paracrine signals with limited regenerative potential.³⁰ Thus, an emerging area of investigation includes developing methods to harness epicardial-derived developmental programs to promote cardiac repair and regeneration in adult hearts. The remainder of this review will highlight what is currently known regarding the role of the epicardium in cardiac regeneration and disease, and how we may take advantage of EPDC plasticity and signaling to promote efficient cardiac repair and remodeling and advance regenerative based medicine for the treatment of human cardiac disease.

• Download figure
• Download PowerPoint

Figure 3. Resident epicardium-derived fibroblasts contribute to fibrosis following myocardial infarction (MI). Evaluation of epicardium-derived cell distribution in left ventricles of mice subjected to sham or myocardial infarction (MI). Epicardial cells are visualized (GFP⁺=green) by crossing constitutive (Wt1 ^CreBAC/+ ) or tamoxifen-inducible (Wt1 ^CreERT2/+ ) Cre lines to the Rosa26 ^mTmG Cre-dependent fluorescent lineage reporter mouse line. A, Epicardial cells of developmental origin (Wt1 ^CreBAC/+ ; Rosa26 ^mTmG/+ ) were observed on the epicardial surface (Epi), and in both perivascular and interstitial areas of the left ventricle of sham-operated hearts. Upon cardiac injury, preexisting GFP⁺ cells contribute to epicardial thickening and the formation of a cellularized scar. Green =GFP⁺ epicardium-derived progenitor cells (EPDCs), red= cardiac troponin T or cTNT, blue= DAPI to visualize nuclei. B, When tamoxifen was administered to 8-week old Wt1 ^CreERT2/+ ; Rosa26 ^mTmG/+ mice, GFP⁺ cells were observed primarily on the epicardial surface of the left ventricle in sham-operated animals. After MI, adult Wt1-lineage derived cells increase in number at the epicardial border surrounding the border zone region of the left ventricle, but they do not directly contribute to scar formation. Green= GFP⁺ EPDCs, red = membrane-tethered tdTomato (nonrecombined cells), blue = DAPI to visualize nuclei. Scale bars for (A) and (B) =50 μm.

Preexisting Fibroblasts Contribute to Cardiac Fibrosis

The developmental origins of a majority of cardiac fibroblasts in the adult heart can be traced back to the fetal epicardium.^39,92 These fibroblasts make up ≈20% of the total composition of cells in the mature mammalian heart,¹²⁵ however, mechanical and ischemic stress leading to the loss of cardiomyocytes and vasculature can increase the total numbers and volume of fibroblasts following cardiac injury. Resident cardiac fibroblasts labeled with Col1a1-GFP, PDGFRα, and Tcf21 transgenic alleles display the ability to proliferate after injury and give rise to a majority of cells in the fibrotic scar.^39,40 Myofibroblasts, labeled by Postn (Periostin), emerge from Tcf21 lineage-traced epicardium-derived fibroblasts when mice are subjected to MI or left ventricle pressure overload and are a major source of ECM.^40,126 Alterations in FN, MAPK (mitogen-activated protein kinase), or Wnt/β-catenin signaling in the Tcf21 lineage significantly alters fibroblast to myofibroblast conversion in response to cardiac stress.^127–129 Interestingly, it was proposed that subpopulations of myofibroblasts re-express Tcf21 following cardiac injury,⁴⁰ which may indicate that a subset of myofibroblasts reverts towards quiescence and senescence in chronic stages of disease. Although preexisting resident cardiac fibroblasts originating from the epicardium during development are the primary source of cardiac scar tissue during ischemic and nonischemic remodeling, nascent EPDCs and resident or infiltrating inflammatory cells have also been observed within the scar tissue, albeit to a lesser extent^32,130 (Figure 3). Tcf21, Tbx18, and Wt1 serve as markers of both developmental and injury-induced epicardial-derived fibroblasts in zebrafish and mammalian adult hearts.^{32,37,40,43,130} In both the human and adult mouse heart, fibroblasts expressing Wt1, Tbx18, and Tcf21 proteins are observed within interstitial fibrosis following ischemic injury.¹³⁰ In contrast, perivascular fibrosis was primarily composed of Tcf21⁺ fibroblasts, but not Wt1⁺ or Tbx18⁺ cells, potentially indicating heterogeneity within epicardial-derived fibroblast lineages. While this study depended on the endogenous protein labeling of epicardial markers, it is interesting to speculate that spatial diversity of epicardial-derived lineages may contribute to functional differences in cardiac repair and remodeling.

In addition to the Tcf21 and Postn lineages, robust investment of Wt1-lineage fibroblasts has been observed in the scar after cardiac injury. Tamoxifen-inducible tracing via Wt1-directed CreERT2 in the adult mouse heart reveals that EPDCs have enriched expression of Snai1, Snai2, and Smad1 and display a propensity towards secretion of proangiogenic factors as compared to non-EPDCs following MI.^30,124 Reactivated Wt1-derived cells acquire a phenotype consistent with fibroblast and myofibroblast lineages as evidenced by the expression of FN, fibroblast-specific protein 1, collagen type III α 1, and smooth muscle markers but are generally devoid of endothelial or cardiomyogenic cell markers.³⁰ Although the contribution of new Wt1⁺ cells towards various intramyocardial fates is limited in the adult, the Wt1 lineage originating during development is the major source of mesenchymal cells within the heart during stress (Figure 3). The constitutive Wt1-Cre line labels >90% of the fibroblasts present in the left ventricular free wall at baseline and following transaortic constriction surgery or MI.^32,33 Additionally, 96% of the Wt1-Cre lineage cells expressed the ECM gene Col1a1.³² Inhibition of embryonic Wt1-Cre lineage mobilization via deletion of Srf or Mrtfs led to a significant reduction in the number of epicardium-derived fibroblasts colonizing the post-MI infarct.¹³¹ Interestingly, the WT1 lineage depleted scar was populated with PDGFRα⁺ cells, apparently from an alternative source.¹³¹

In complementary lineage-tracing experiments, resident fibroblast composition within the uninjured heart is ≈75% Tbx18⁺ and 90% Wt1⁺.^32,33,37 Tbx18⁺/Thy1 cell surface antigen⁺ fibroblasts are dispersed throughout the heart and express markers of fibroblasts, SMCs, and cardiac pericytes,³⁷ whereas Wt1-lineage cells are primarily localized to the ventricular septum and left ventricle free wall.^32,33 Although injury typically increases the total number of fibroblasts in the heart due to proliferation, the percentage of either Tbx18 or Wt1-derived fibroblasts was unaltered following transaortic constriction surgery.^32,37 These data may be interpreted to indicate resident fibroblasts may arise from the heterogeneous compilation of various developmental epicardial lineages.

Mesenchymal cell derivatives, such as cardiac pericytes, have also been implicated in fibrotic diseases. However, Tbx18-lineage derived cardiac pericytes do not exhibit mesenchymal cell characteristics in vivo after transaortic constriction surgery or prolonged aging in mice.³⁴ Due to these inconsistencies, further investigation is required to rule out a potential role of pericytes in adult cardiac repair processes, particularly in vascular remodeling during ischemic events. Taken together, these studies also conclude that epicardial activation does not contribute to the formation of de novo fibroblasts during adult pressure overload or following MI. Overall, understanding the cellular origin and heterogeneity of cardiac mesenchymal cells may offer critical insights for ameliorating the proliferation and differentiation of pathological myofibroblasts during the progression of heart disease.

The Epicardium Modulates Inflammation

Acute inflammation is a compensatory response that clears tissue of dead cells; however, chronic inflammation can increase cell death, formation of fibrosis, and cardiac dysfunction.¹³² Recent reports suggest that cellular and paracrine inflammatory processes are, in part, coordinated by the reactivated epicardium.^117,133 CD45⁺ hematopoietic cells emerge during fetal development alongside Wt1⁺ EPDCs, and both become activated and migrate towards the subepicardium upon cardiac injury.¹³⁴ Resident cardiac macrophages with reparative characteristics (M2 macrophages) are observed in the healthy epicardium but are significantly depleted in models of aging-related cardiac dysfunction.¹³⁵ Wt1b is an early epicardial response gene that overlaps with expression of proregenerative macrophages in zebrafish¹³⁶ implicating the requirement of the epicardium in the recruitment of cardiac macrophages as observed during development.¹¹⁷ Indeed, epicardial deficiency in YAP (yes associated protein 1)/TAZ (WW domain containing transcription regulator 1) signaling exacerbates cardiac inflammation after MI due to the limited recruitment of immunosuppressive regulatory T cells and depressed secretion of inflammatory cytokine interferon-γ from the adult mouse epicardium.¹³⁷ Attempts to modulate epicardial activation have harnessed the activity of CCAAT/enhancer-binding proteins, a group of TFs that bind to enhancer elements controlling epicardial gene expression.¹³³ Adenoviral delivery of CCAAT/enhancer-binding protein following ischemia/reperfusion injury in mice enhances activation of Wt1 and Raldh2, blunting cardiac damage and the initiation of fibrosis, presumably through decreased acute neutrophil infiltration.¹³³ These studies emphasize the importance of epicardium reactivation in inflammatory signaling and the promise of harnessing epicardial cell biology for cardiac repair.

Epicardium Contribution to Subepicardial Adipose Tissue

The human heart is surrounded by a layer fat called epicardial adipose tissue (EAT) that lies beneath the epicardium. The extent of EAT has prognostic value and correlates with various pathological conditions such as coronary artery disease and ischemic remodeling.¹³⁸ While a better understanding of the developmental source and molecular mechanisms that control EAT accumulation may have therapeutic implications, the study of EAT biology has been hampered by its paucity in experimental model organisms. Advances in genetic lineage tracing in mice have recently revealed that EAT originates from the epicardium in a process mediated by the metabolic regulator peroxisome proliferator-activated receptor-γ.¹³⁹ Furthermore, delivery of IGF-1 to mice augments epicardial cell conversion into adipocytes in experimental MI.²⁸

Importantly, disorders such as arrhythmogenic right ventricular cardiomyopathy are characterized by fibrofatty tissue deposition that replaces functional heart muscle and markedly disturbs cardiac conduction in human patients.^140–142 Recent evidence suggests that mutations in the plakophilin-2 gene are causative in arrhythmogenic right ventricular cardiomyopathy through disruption of the cardiomyocyte desmosome.¹⁴³ In addition, plakophilin-2 may modulate epicardial cell migration and initiate an adipogenic program in epicardial cells, potentially relevant due to the epicardium-to-endocardium gradient of fibrofatty tissue deposition.¹⁴⁴ Defining the cellular and molecular avenues leading to fibrosis and the formation of EAT may thus lead to new clinical treatment options to prevent progression of arrhythmogenic cardiomyopathies. Collectively, reactivation of the adult heart due to cardiac disease encourages cell contributions and paracrine signals from the epicardium to take part in fibrotic, adipogenic, and vasculature remodeling, cardiomyocyte growth and cell death, and inflammatory signaling (Figure 4). Harnessing the regenerative power of the fetal epicardium at the expense of insult-responsive pathological programs holds promise for the advancement of new cardiac restoration strategies, as described in regenerative models below.

• Download figure
• Download PowerPoint

Figure 4. Epicardium-derived cells in the cardiac injury response. Preexisting epicardium-derived resident cardiac fibroblasts are the primary source of myofibroblasts (MyoFbs) that generate scar tissue during pressure overload or ischemic remodeling through the secretion ECM (extracellular matrix). Epicardium-derived pericytes (PC) or endothelial cells (EC) have also been reported to exhibit mesenchymal cell characteristics and produce ECM in response to injury. The adult epicardium is reactivated and expands in response to cardiac injury. A small proportion of epicardium-derived progenitor cells (EPDCs) undergo epithelial-to-mesenchymal transition (EMT) to become MyoFbs that secrete extracellular matrix (ECM). In limited cases, EPDCs can become adipocytes (AC). The activated epicardium also promotes the recruitment of inflammatory cells (Neutrophils or NT, T-regulatory cells or Treg⁺, and Macrophages or MC) and stabilizes coronary vasculature through the secretion of chemokines (dashed arrows). The epicardium may also promote cardiomyocyte (CM) survival and/or cell growth. Epi indicates epicardium; myo, myocardium; and subepi, subepicardium. Illustration credit: Ben Smith.

Mechanisms of Cardiac Regeneration

Many adult amphibians and fish possess an incredible capacity to completely regenerate damaged tissue, including the heart.¹²³ Tissue repair in axolotl and zebrafish is initiated by the migration of multipotent mesenchymal cells to the wound and the formation a regenerative blastema.⁶ Particularly in zebrafish hearts, surgical removal of ≈10% to 15% of the cardiac apex results in the formation of a reversible fibrin clot followed by the remarkable restoration of cardiac muscle via the proliferation of preexisting cardiomyocytes.¹⁴⁵ Zebrafish heart regeneration occurs efficiently due to 4 essential processes: (1) efficient cardiomyocyte proliferation, (2) endocardium activation, (3) reactivation of epicardial fetal gene expression, and (4) efficient resolution of fibrosis.^145,146 In contrast, the adult mammalian heart is unable to regenerate following injury; however, neonatal mammals retain the capacity to regenerate cardiac tissue following apical resection, cryoinjury, or MI when the injury is performed before 7 days after birth, which is within the window when preexisting cardiomyocyte proliferation can be stimulated.^147,148 Nonmyocytes, including epicardial cells, are reported to play critical roles in cardiac regeneration by regulating inflammation, stimulating cardiomyocyte proliferation, and modulating ECM turnover.¹⁴⁹

The ECM Landscape in Regeneration

ECM modulates the fate and function of EPDCs in the embryo or during regenerative cardiac repair; and likewise, epicardial cells provide a unique extracellular milieu that mediates cardiac healing. In the healthy adult mouse heart, clusters of 2 or more Wt1⁺ cells are encased within ECM components, such as FN, collagen IV, and hyaluronan, which help to define an epicardial-specific niche.¹³⁴ In zebrafish, the epicardium supports cardiomyocyte recruitment to sites of injury via the secretion of fn1 and fn1b, which presumably bind the FN receptor (integrinb3) on cardiomyocytes and promote motility required for regeneration.¹⁵⁰ In addition to FN, hyaluronan can also promote regeneration. Hyaluronan signaling through the Hmmr (hyaluronan-mediated motility receptor) is induced 3 days post resection of the zebrafish heart; blocking Hmmr or inhibiting hyaluronan synthesis suppresses cardiomyocyte proliferation and exacerbates scar formation.¹⁵¹ Importantly, inhibition of the hyaluronan-Hmmr axis perturbs cell-to-cell anchoring via focal adhesion kinase, which limits the expression of EMT genes snail1a, snail1b, snail2, and twist1.¹⁵¹ Collectively, these data demonstrate that epicardial ECM production and EMT are essential in processes of adult cardiac regeneration.

Paracrine Regulation of Zebrafish Cardiac Regeneration

Intercellular signaling between the myocardium, epicardium, fibroblasts, and endocardium coordinate regenerative processes in the adult zebrafish heart. PDGF, FGF, IGF, and VEGF ligands influence cardiomyocyte growth and proliferation in development and are proposed to promote cardiomyocyte dedifferentiation and the upregulation of cell cycle regulators during regeneration.^152–155 Disruption of the epicardium can lead to a marked reduction in regenerative programs consistent with the requirement of epicardial-secreted factors in cardiomyocyte proliferation. Based on initial microarray analysis of the early zebrafish cardiac wound, pdgfa and pdgfb genes are both highly expressed in regenerating tissue.¹⁵⁶ Administration of PDGF-BB is sufficient to induce cardiomyocyte proliferation, whereas chemical inhibition of PDGF prevents cardiomyocyte DNA synthesis in culture.¹⁵⁶ Zebrafish hearts exhibit an upregulation of classical EMT regulators (snai2 and twist1b) that correlate with induction of the mural cell marker gene, Pdgfrβ, while Pdgfrα, a fibroblast marker, was unchanged.¹⁵⁵ Inhibition of PDGFR following injury limits epicardial proliferation in vivo and further suppresses the expression of EMT (snai2) and mural cell (acta2) markers.¹⁵⁶ Notably, PDGFR inhibition leads to impaired coronary vessel formation signifying the requirement of PDGF signaling in the process of revascularization and regeneration in the zebrafish heart.¹⁵⁵

FGF signaling functions downstream of RA signaling and is necessary for fin repair and cardiac regeneration in zebrafish.^153,157 Global deletion of fgfr1 results in a reduction of Tbx18⁺ EPDCs in the ventricle and correlates with increased incidence of cardiomyocyte hypoplasia.¹⁵⁷ Following acute injury, fgfr2 and fgfr4 are increased in EPDCs surrounding the atria, whereas fgfr4 is expressed adjacent to the wound and up to 30-day post resection.¹⁵³ Suppression of FGFR signaling in the epicardium disrupts EMT and neovascularization of damaged tissue.¹⁵³

IGF was discovered as a potent mitogen during cardiac development.¹¹⁰ Activated igf2b is observed in the endocardium and epicardial surface and surrounding apical wounds of the adult injured zebrafish heart.^133,154 Limiting igf2 or igf1r suppresses DNA synthesis in cardiomyocytes following resection.¹⁵⁴ Further examination of cardiomyocyte proliferation using the gata4:EGFP reporter confirms that IGF signaling is required for the proliferation of subepicardial cardiomyocytes during wound repair.¹⁵⁴ Notch receptor (notch1a, notch2, and notch3) expression is upregulated in both the epicardium and endocardium; Notch activity is required for regenerative replacement of cardiomyocytes but is dispensable for the formation of primitive endothelial tubes in the wound area.¹⁵⁸ The epicardium is also a source of soluble factors that control cardiomyocyte and vasculature regeneration through the secretion of vegfaa.¹⁵² Interestingly, PDGF, FGF, and VEGF ligands interact with neuropilin transmembrane receptors, which are activated in response to cryoinjury in both endocardial and epicardial cells.¹⁵⁹ Loss-of-function of neuropilin1a significantly impairs Wt1-induced epicardial reactivation and EMT following cryoinjury.¹⁵⁹

Of note, the epicardium itself has a regenerative capacity. Deletion of the epicardium in the adult zebrafish resection model significantly reduces cardiomyocyte proliferation and regeneration.¹⁴⁹ Hedgehog signaling, expressed primarily in the bulbus arteriosus (analogous to the mouse outflow tract), is required for epicardial activation and regrowth.¹⁴⁹ Indeed, removal of the bulbous arteriosus impairs epicardial activation and migration towards the ventricle, as well as cardiac regeneration after injury, an outcome that can be reversed after implantation of Sonic hedgehog soaked beads in the ventricle.¹⁴⁹ Overall, studies directed at identifying the instructive cues to support the replenishment and recruitment of epicardial cells may lead to novel therapeutic strategies to enhance epicardium-mediated cardiac repair.

Epicardial-Derived Lineages in Zebrafish Regeneration

In zebrafish, tcf21 is highly expressed in epicardial precursors that give rise to perivascular cells during development and regeneration.⁴³ Genetic ablation of tcf21 ⁺ EPDCs results in prolonged fibrin clot deposition and collagen expression negatively impacting cardiomyocyte turnover and revascularization after injury.¹⁴⁹ However, even in cases where 80% of Tcf21⁺ cells are depleted from the epicardium, Tcf21⁺ cells repopulate the surface of the heart within 60 days post amputation indicating the endogenous reparative capacity of the epicardium.¹⁴⁹ Changes in mechanical tension have been recently shown to facilitate regeneration of the epicardium following ablation.¹⁶⁰ Using a strategy to deplete tcf21 in zebrafish,¹⁴⁹ the migratory edge of the regenerating epicardium was populated by hypertrophic and multinucleated leader cells, which subsequently underwent apoptosis allowing for the persistence of mononucleated epicardial cells in regenerative tissue.¹⁶⁰ This study also indicated that mechanical signaling can regulate epicardial regeneration, such as through modification of cellular behavior, including cell death, nuclear polarity, and migration; all factors that may significantly alter cellular processes of differentiation and secretion during cardiac repair.

Pan-epicardial reporters wt1a and wt1b have further revealed that the epicardium contributes to fibroblast plasticity in zebrafish heart regeneration.¹⁴⁶ The wt1b lineage reporter labels cells present at the epicardial border that contribute to fibroblastic collagen1a2 ⁺ and postn ⁺ cells in response to cryoinjury.¹⁴⁶ Additionally, wt1b ⁺;postn ⁺ cells revert towards a quiescent state as evidenced by the noticeable downregulation of ECM genes, a process that appears to be conducive to cardiomyocyte proliferation.¹⁴⁶ This transient fibrosis mediated by cardiac fibroblast plasticity should be further examined to identify mechanisms that might lead to resolution of fibrosis during cardiac remodeling in adult mammalian models. In summary, the combination of in vivo and in vitro studies presented here contribute to the understanding of EPDC responsiveness and secretion of growth factors that impact potential downstream signaling programs associated with enhanced cardiac regeneration (Figure 5).

• Download figure
• Download PowerPoint

Figure 5. Epicardium in cardiac regeneration. Cardiac regeneration occurs in response to a number of stimuli including apical resection, neonatal myocardial infarction, cryoablation, or targeted ablation of the epicardium in both zebrafish and the early postnatal mammalian heart. The epicardium actively regenerates through the migration of leader binucleated epicardial cells initiating wound closure and contributes to the formation of perivascular cells (smooth muscle cells [SMCs] and fibroblasts [Fbs]). Epicardium-derived fibroblasts have been shown to acquire myofibroblast (MyoFb) phenotypes (MyoFb and extracellular matrix [ECM]), which may be reversible during the resolution phase of the scar. The epicardium promotes macrophage (MC) recruitment, neovascularization as well as cardiomyocyte (CM) cell cycle re-entry via secretion of mitogens (dashed arrows). epi indicates epicardium; myo, myocardium; and subepi, subepicardium. Illustration credit: Ben Smith.

Mechanisms of Postnatal Heart Regeneration

Neonatal mammalian cardiomyocytes demonstrate preserved cell cycle activity underlying the endogenous regenerative capacity of the mouse heart in the first week of life.¹⁴⁷ Apical resection (≈15% of the ventricle) or permanent ligation induced-MI at postnatal day 1 to 2 results in complete regeneration by 3 weeks post injury due to increased cardiomyocyte proliferation.¹⁴⁷ Like zebrafish, acute upregulation of Wt1 and EMT marker genes are detected following apical resection and cryoinjury, as well as permanent coronary artery ligation in the neonatal mouse heart.^147,161 More recently, Tbx18⁺ cell accumulation was observed in the neonatal heart following apical resection; however, Tbx18 lineage-traced cells display minimal differentiation potential.¹⁶² Despite the limited regenerative capacity of the adult EPDCs, recent studies have been aimed at discovering molecular and cellular programs that enhance and extend regeneration beyond the early postnatal window by preventing cardiomyocyte cell cycle arrest and limiting fibroblast activation. Previous reports in development show that FGF-10 is critical for cardiomyocyte maturation,¹⁰⁹ and although FGF-10 treatment promoted the expansion of epicardial cells in the neonatal heart, FGF treatment did not significantly enhance cardiac regeneration.¹⁶³ However, pretreatment of neonatal cardiac tissue with thymosin-β4, which has been previously shown to enhance revascularization and cardiomyogenesis in the adult,^164,165 stimulated regeneration following resection at 7 days post birth, mediated, in part, by the enhanced migration and differentiation of Wt1⁺ cells in the wound.¹⁶⁶ In addition to defining factors that have effective and combinatorial influence on cardiomyocyte proliferation and the prevention of fibrosis, stimulation of neoangiogenesis during neonatal cardiac regeneration has been largely overlooked. We previously described the importance of the positional cues stemming from the epicardium that can regulate the formation of the coronary vasculature.^120,121 Similar to studies observed during cardiac development, efficient neonatal regeneration is prevented in models exhibiting Cxcl12 deletion.¹⁶⁷ Exogenous CXCL12 administration facilitated arterial cell reorganization and collateral artery formation 14 days post-MI in the adult mouse heart, although adult cardiac physiology was not evaluated in this study.¹⁶⁷

Recruitment of inflammatory cells may also play a critical role in stimulation of angiogenesis and scar resolution in neonatal heart regeneration. Clodronate liposome-mediated depletion of monocytes and macrophages during the postnatal regenerative window augments fibrotic remodeling and impairs neovascularization in both peri-infarct and distal regions.¹⁶⁸ Transcriptional analysis of macrophages between postnatal day 1 and day 21 suggests that loss of regenerative potential with time is associated with macrophage polarization and expression of anti-inflammatory factors.¹⁶⁸ However, these studies suggest that in addition to immune cells, a combination of cardiac cells, including ECs and epicardial cells, likely contribute secretory factors required for cardiac repair. Here, we presented some of the factors that may be harnessed to prolong the postnatal regenerative window, which may be adapted to induce cycling of adult cardiomyocytes and stimulation of angiogenic processes that are needed to promote repair in human diseased hearts.

Future Perspectives

In this review, we have discussed the gene programs that regulate cardiac growth and tissue repair through both cell autonomous and cell nonautonomous functions of the epicardium. Our broad analysis describes the study of epicardial-secreted factors, such as PDGFs, IGFs, or thymosin-β4, and the modulation of inflammatory factors that might enhance epicardial mediated cardiac repair.^92,111,165 Additionally, the development of therapeutic strategies that re-direct EPDCs towards proregenerative cells types at the expense of fibroblast and adipocyte differentiation could complement current regenerative medicine approaches that are largely based on stem and induced pluripotent cell delivery.

Enhancement of endogenous cardiac repair via the isolation, expansion, and transplantation of progenitor cells derived from bone marrow and cardiac niches have seen limited success. Methods to derive primary EPDCs from zebrafish, mouse, and human hearts have been validated and may lead to the creation of novel progenitor cell sources for therapies in the future.^87,169,170 Adult human EPDCs recapitulate EMT-like processes of fetal human EPDCs, including the ability to migrate and differentiate into mural cell lineages.¹⁶⁹ However, the promise of cell transplantation for regenerative purposes can be hampered by immune rejection if the donor is not autologous to the recipient. Recent advances in human induced pluripotent stem cell technologies have demonstrated the feasibility of differentiating pluripotent cells into epicardial cells and their derivatives through the modulation of bone morphogenic factor, Wnt, FGF, and RA signaling pathways.¹⁷¹ Co-culture of human embryonic stem cell–derived epicardial cells (hESC-EPDCs) with hESC-derived cardiomyocytes (hESC-CMs) was shown to promote cardiac contractility and cardiomyocyte maturation in engineered heart tissues.¹⁷² Furthermore, co-transplantation of hESC-EPDCs and hESC-CMs into infarcted rat hearts enhances engraftment, improves systolic function, and augments revascularization compared with hESC-CMs alone,¹⁷² making promising strides towards therapeutic practice.

Recent developments in evaluating epicardial activation and lineage commitment in various cardiac disease models indicate that cardiac regeneration is enhanced by facilitating crosstalk between the epicardium and ECs or cardiomyocytes. Replicating endogenous cross talk was accomplished via epicardial cell-conditioned media delivery to the surface of the heart in a 3-dimensional collagen patch, which promotes cardiomyocyte maturation and improves cardiac function after MI.¹⁷³ Furthermore, this study identified FSTL-1 (Follistatin-like 1) as the primary and potent epicardial cardiogenic factor to enhance cardiac function and repair after patch implementation.¹⁷³ FSTL-1 treatment results in reduced fibrosis, improved revascularization and increased cardiomyocyte proliferation in both adult mouse and porcine preclinical models indicating an applicability of epicardial-derived paracrine factors in future clinical practices.¹⁷³ Cell-free therapeutics, such as synthetic modified RNA and small molecules, can promote the expression of proregenerative genes in multiple cell types including the epicardium.^28,164 Delivery of a modified RNA expressing VEGF-A activates EPDCs and promotes their migration and differentiation towards EC fate after cardiac injury.¹⁷⁴ Therefore, as mentioned throughout this review, knowledge of upstream growth factor signaling can be used to enhance epicardial cell expansion in vitro or the generation of novel therapeutic strategies for clinical practice.

Finally, important questions remain regarding epicardial cell heterogeneity, which may complicate strategies to harness the epicardium for cardiac repair or may lead to identification of rare or unique EPDCs that are particularly amenable to proregenerative approaches. Transcriptional profiling of the epicardium using bulk and single-cell RNA-sequencing platforms (RNA-seq or scRNA-seq, respectively) can prove useful in identifying genes and pathways that respond to cardiac injury. RNA-seq performed on laser captured epicardial tissue identified many markers that distinguish noninjured and injured epicardial cells.⁵⁵ In zebrafish, the Tcf21 lineage appears to be comprised of a number of distinct epicardium-derived cell populations.¹⁷⁵ This study reveals the complex heterogeneity within the Tcf21 lineage and encourages the use of single-cell transcriptomics to study progenitor cell biology effectively in various model organisms and patient samples. A more comprehensive analysis of epicardial cell contributions to normal development, congenital cardiomyopathy, and heart failure and may reveal unanticipated mechanisms that can be harnessed to improve cardiac regeneration and repair.

Nonstandard Abbreviations and Acronyms

Col1a1	collagen1a1
CXCL	C-X-C motif chemokine receptor ligand
CXCR	CXC chemokines and receptor
EAT	epicardial adipose tissue
EC	endothelial cell
ECM	extracellular matrix
EMT	epithelial-to-mesenchymal transition
EPDC	epicardium-derived progenitor cell
FGF	fibroblast growth factor
FGFR	fibroblast growth factor receptor
FN	fibronectin
FSTL-1	Follistatin-like 1
hESC	human embryonic stem cell
Hmmr	hyaluronan-mediated motility receptor
IGF	insulin growth factor
IR	ischemia/reperfusion
MAPK	mitogen-activated protein kinase
MI	myocardial infarction
MRTF	myocardin-related transcription factor
PDGF	platelet-derived growth factor
PDGFR	platelet-derived growth factor receptor
Postn	periostin
RA	retinoic acid
Raldh	retinaldehyde dehydrogenase
RXR	retinoid x receptor
Scx	scleraxis
SMC	smooth muscle cell
SRF	serum-response factor
TAZ	WW domain-containing transcription regulator 1
Tbx18	T-box protein 18
TF	transcription factor
TGF-β	transforming growth factor β
VEGF	vascular endothelial growth factor
WT1	Wilms tumor-1
YAP	yes associated protein 1

Sources of Funding

Generous grant funding was provided to P. Quijada (NIHT32HL066988, NIHF32HL134206, and the American Heart Association 19CDA34590003); M.A. Trembley (T32HL07572, NIHT32HL066988, NIHT32GM068411, the American Heart Association 16PRE3049000, and the Howard Hughes Medical Institute Med-Into-Grad program); and E.M. Small (NIHR01-HL133761, NIHR01-HL136179, NIHR01-HL120919, NIHR01-HL144867, NIHUL1-TR002001, and NYSTEM-C32566GG).

Footnotes

For Sources of Funding and Disclosures, see page 390.

Correspondence to: Eric M. Small, PhD, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Box CVRI, Rochester, NY 14642. Email [email protected] rochester.edu

References

• 1. Benjamin EJ, Muntner P, Alonso A, Bittencourt MS, Callaway CW, Carson AP, Chamberlain AM, Chang AR, Cheng S, Das SR, et al. ; American Heart Association Council on Epidemiology and Prevention Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics-2019 update: a report from the American Heart Association. Circulation . 2019; 139:e56–e528. doi: 10.1161/CIR.0000000000000659LinkGoogle Scholar
• 2. Burchfield JS, Xie M, Hill JA. Pathological ventricular remodeling: mechanisms: part 1 of 2. Circulation . 2013; 128:388–400. doi: 10.1161/CIRCULATIONAHA.113.001878LinkGoogle Scholar
• 3. Heallen TR, Kadow ZA, Kim JH, Wang J, Martin JF. Stimulating cardiogenesis as a treatment for heart failure. Circ Res . 2019; 124:1647–1657. doi: 10.1161/CIRCRESAHA.118.313573LinkGoogle Scholar
• 4. Vagnozzi RJ, Molkentin JD, Houser SR. New myocyte formation in the adult heart: endogenous sources and therapeutic implications. Circ Res . 2018; 123:159–176. doi: 10.1161/CIRCRESAHA.118.311208LinkGoogle Scholar
• 5. Forte E, Furtado MB, Rosenthal N. The interstitium in cardiac repair: role of the immune-stromal cell interplay. Nat Rev Cardiol . 2018; 15:601–616. doi: 10.1038/s41569-018-0077-xCrossrefMedlineGoogle Scholar
• 6. Cao J, Poss KD. The epicardium as a hub for heart regeneration. Nat Rev Cardiol . 2018; 15:631–647. doi: 10.1038/s41569-018-0046-4CrossrefMedlineGoogle Scholar
• 7. Meilhac SM, Lescroart F, Blanpain C, Buckingham ME. Cardiac cell lineages that form the heart. Cold Spring Harb Perspect Med . 2015; 5:a026344. doi: 10.1101/cshperspect.a026344CrossrefMedlineGoogle Scholar
• 8. Risebro CA, Vieira JM, Klotz L, Riley PR. Characterisation of the human embryonic and foetal epicardium during heart development. Development . 2015; 142:3630–3636. doi: 10.1242/dev.127621CrossrefMedlineGoogle Scholar
• 9. Pombal MA, Carmona R, Megías M, Ruiz A, Pérez-Pomares JM, Muñoz-Chápuli R. Epicardial development in lamprey supports an evolutionary origin of the vertebrate epicardium from an ancestral pronephric external glomerulus. Evol Dev . 2008; 10:210–216. doi: 10.1111/j.1525-142X.2008.00228.xCrossrefMedlineGoogle Scholar
• 10. Serluca FC. Development of the proepicardial organ in the zebrafish. Dev Biol . 2008; 315:18–27. doi: 10.1016/j.ydbio.2007.10.007CrossrefMedlineGoogle Scholar
• 11. Nahirney PC, Mikawa T, Fischman DA. Evidence for an extracellular matrix bridge guiding proepicardial cell migration to the myocardium of chick embryos. Dev Dyn . 2003; 227:511–523. doi: 10.1002/dvdy.10335CrossrefMedlineGoogle Scholar
• 12. Zhou B, von Gise A, Ma Q, Rivera-Feliciano J, Pu WT. Nkx2-5- and Isl1-expressing cardiac progenitors contribute to proepicardium. Biochem Biophys Res Commun . 2008; 375:450–453. doi: 10.1016/j.bbrc.2008.08.044CrossrefMedlineGoogle Scholar
• 13. Tandon P, Miteva YV, Kuchenbrod LM, Cristea IM, Conlon FL. Tcf21 regulates the specification and maturation of proepicardial cells. Development . 2013; 140:2409–2421. doi: 10.1242/dev.093385CrossrefMedlineGoogle Scholar
• 14. Rodgers LS, Lalani S, Runyan RB, Camenisch TD. Differential growth and multicellular villi direct proepicardial translocation to the developing mouse heart. Dev Dyn . 2008; 237:145–152. doi: 10.1002/dvdy.21378CrossrefMedlineGoogle Scholar
• 15. Zhou B, Ma Q, Rajagopal S, Wu SM, Domian I, Rivera-Feliciano J, Jiang D, von Gise A, Ikeda S, Chien KR, et al. . Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature . 2008; 454:109–113. doi: 10.1038/nature07060CrossrefMedlineGoogle Scholar
• 16. Carmona R, Guadix JA, Cano E, Ruiz-Villalba A, Portillo-Sánchez V, Pérez-Pomares JM, Muñoz-Chápuli R. The embryonic epicardium: an essential element of cardiac development. J Cell Mol Med . 2010; 14:2066–2072. doi: 10.1111/j.1582-4934.2010.01088.xCrossrefMedlineGoogle Scholar
• 17. Zamora M, Männer J, Ruiz-Lozano P. Epicardium-derived progenitor cells require beta-catenin for coronary artery formation. Proc Natl Acad Sci U S A . 2007; 104:18109–18114. doi: 10.1073/pnas.0702415104CrossrefMedlineGoogle Scholar
• 18. Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol . 2014; 15:178–196. doi: 10.1038/nrm3758CrossrefMedlineGoogle Scholar
• 19. Mikawa T, Gourdie RG. Pericardial mesoderm generates a population of coronary smooth muscle cells migrating into the heart along with ingrowth of the epicardial organ. Dev Biol . 1996; 174:221–232. doi: 10.1006/dbio.1996.0068CrossrefMedlineGoogle Scholar
• 20. Dettman RW, Denetclaw W, Ordahl CP, Bristow J. Common epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart. Dev Biol . 1998; 193:169–181. doi: 10.1006/dbio.1997.8801CrossrefMedlineGoogle Scholar
• 21. Pérez-Pomares JM, Phelps A, Sedmerova M, Carmona R, González-Iriarte M, Muñoz-Chápuli R, Wessels A. Experimental studies on the spatiotemporal expression of WT1 and RALDH2 in the embryonic avian heart: a model for the regulation of myocardial and valvuloseptal development by epicardially derived cells (EPDCs). Dev Biol . 2002; 247:307–326. doi: 10.1006/dbio.2002.0706CrossrefMedlineGoogle Scholar
• 22. Braitsch CM, Yutzey KE. Transcriptional control of cell lineage development in epicardium-derived cells. J Dev Biol . 2013; 1:92–111. doi: 10.3390/jdb1020092CrossrefMedlineGoogle Scholar
• 23. Cano E, Carmona R, Ruiz-Villalba A, Rojas A, Chau YY, Wagner KD, Wagner N, Hastie ND, Muñoz-Chápuli R, Pérez-Pomares JM. Extracardiac septum transversum/proepicardial endothelial cells pattern embryonic coronary arterio-venous connections. Proc Natl Acad Sci U S A . 2016; 113:656–661. doi: 10.1073/pnas.1509834113CrossrefMedlineGoogle Scholar
• 24. Merki E, Zamora M, Raya A, Kawakami Y, Wang J, Zhang X, Burch J, Kubalak SW, Kaliman P, Izpisua Belmonte JC, et al. . Epicardial retinoid X receptor alpha is required for myocardial growth and coronary artery formation. Proc Natl Acad Sci U S A . 2005; 102:18455–18460. doi: 10.1073/pnas.0504343102CrossrefMedlineGoogle Scholar
• 25. Trembley MA, Velasquez LS, de Mesy Bentley KL, Small EM. Myocardin-related transcription factors control the motility of epicardium-derived cells and the maturation of coronary vessels. Development . 2015; 142:21–30. doi: 10.1242/dev.116418CrossrefMedlineGoogle Scholar
• 26. Zhou B, von Gise A, Ma Q, Hu YW, Pu WT. Genetic fate mapping demonstrates contribution of epicardium-derived cells to the annulus fibrosis of the mammalian heart. Dev Biol . 2010; 338:251–261. doi: 10.1016/j.ydbio.2009.12.007CrossrefMedlineGoogle Scholar
• 27. Zhou B, Pu WT. Genetic Cre-loxP assessment of epicardial cell fate using Wt1-driven Cre alleles. Circ Res . 2012; 111:e276–e280. doi: 10.1161/CIRCRESAHA.112.275784LinkGoogle Scholar
• 28. Zangi L, Oliveira MS, Ye LY, Ma Q, Sultana N, Hadas Y, Chepurko E, Später D, Zhou B, Chew WL, et al. . Insulin-like growth factor 1 receptor-dependent pathway drives epicardial adipose tissue formation after myocardial injury. Circulation . 2017; 135:59–72. doi: 10.1161/CIRCULATIONAHA.116.022064LinkGoogle Scholar
• 29. Liu Q, Huang X, Oh JH, Lin RZ, Duan S, Yu Y, Yang R, Qiu J, Melero-Martin JM, Pu WT, et al. . Epicardium-to-fat transition in injured heart. Cell Res . 2014; 24:1367–1369. doi: 10.1038/cr.2014.125CrossrefMedlineGoogle Scholar
• 30. Zhou B, Honor LB, He H, Ma Q, Oh JH, Butterfield C, Lin RZ, Melero-Martin JM, Dolmatova E, Duffy HS, et al. . Adult mouse epicardium modulates myocardial injury by secreting paracrine factors. J Clin Invest . 2011; 121:1894–1904. doi: 10.1172/JCI45529CrossrefMedlineGoogle Scholar
• 31. Wessels A, van den Hoff MJ, Adamo RF, Phelps AL, Lockhart MM, Sauls K, Briggs LE, Norris RA, van Wijk B, Perez-Pomares JM, et al. . Epicardially derived fibroblasts preferentially contribute to the parietal leaflets of the atrioventricular valves in the murine heart. Dev Biol . 2012; 366:111–124. doi: 10.1016/j.ydbio.2012.04.020CrossrefMedlineGoogle Scholar
• 32. Moore-Morris T, Guimarães-Camboa N, Banerjee I, Zambon AC, Kisseleva T, Velayoudon A, Stallcup WB, Gu Y, Dalton ND, Cedenilla M, et al. . Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis. J Clin Invest . 2014; 124:2921–2934. doi: 10.1172/JCI74783CrossrefMedlineGoogle Scholar
• 33. Moore-Morris T, Cattaneo P, Guimarães-Camboa N, Bogomolovas J, Cedenilla M, Banerjee I, Ricote M, Kisseleva T, Zhang L, Gu Y, et al. . Infarct fibroblasts do not derive from bone marrow lineages. Circ Res . 2018; 122:583–590. doi: 10.1161/CIRCRESAHA.117.311490LinkGoogle Scholar
• 34. Guimaraes-Camboa N, Cattaneo P, Sun Y, Moore-Morris T, Gu Y, Dalton ND, Rockenstein E, Masliah E, Peterson KL, Stallcup WB, et al. . Pericytes of multiple organs do not behave as mesenchymal stem cells in vivo. Cell Stem Cell . 2017; 20:345–359.e5. doi: 10.1016/j.stem.2016.12.006CrossrefMedlineGoogle Scholar
• 35. Cai CL, Martin JC, Sun Y, Cui L, Wang L, Ouyang K, Yang L, Bu L, Liang X, Zhang X, et al. . A myocardial lineage derives from Tbx18 epicardial cells. Nature . 2008; 454:104–108. doi: 10.1038/nature06969CrossrefMedlineGoogle Scholar
• 36. Volz KS, Jacobs AH, Chen HI, Poduri A, McKay AS, Riordan DP, Kofler N, Kitajewski J, Weissman I, Red-Horse K. Pericytes are progenitors for coronary artery smooth muscle. Elife . 2015; 4:e10036. doi: 10.7554/eLife.10036CrossrefMedlineGoogle Scholar
• 37. Ali SR, Ranjbarvaziri S, Talkhabi M, Zhao P, Subat A, Hojjat A, Kamran P, Müller AM, Volz KS, Tang Z, et al. . Developmental heterogeneity of cardiac fibroblasts does not predict pathological proliferation and activation. Circ Res . 2014; 115:625–635. doi: 10.1161/CIRCRESAHA.115.303794LinkGoogle Scholar
• 38. Acharya A, Baek ST, Banfi S, Eskiocak B, Tallquist MD. Efficient inducible Cre-mediated recombination in Tcf21 cell lineages in the heart and kidney. Genesis . 2011; 49:870–877. doi: 10.1002/dvg.20750CrossrefMedlineGoogle Scholar
• 39. Acharya A, Baek ST, Huang G, Eskiocak B, Goetsch S, Sung CY, Banfi S, Sauer MF, Olsen GS, Duffield JS, et al. . The bHLH transcription factor Tcf21 is required for lineage-specific EMT of cardiac fibroblast progenitors. Development . 2012; 139:2139–2149. doi: 10.1242/dev.079970CrossrefMedlineGoogle Scholar
• 40. Kanisicak O, Khalil H, Ivey MJ, Karch J, Maliken BD, Correll RN, Brody MJ, J Lin SC, Aronow BJ, Tallquist MD, et al. . Genetic lineage tracing defines myofibroblast origin and function in the injured heart. Nat Commun . 2016; 7:12260. doi: 10.1038/ncomms12260CrossrefMedlineGoogle Scholar
• 41. Quaggin SE, Vanden Heuvel GB, Igarashi P. Pod-1, a mesoderm-specific basic-helix-loop-helix protein expressed in mesenchymal and glomerular epithelial cells in the developing kidney. Mech Dev . 1998; 71:37–48. doi: 10.1016/s0925-4773(97)00201-3CrossrefMedlineGoogle Scholar
• 42. Lu J, Chang P, Richardson JA, Gan L, Weiler H, Olson EN. The basic helix-loop-helix transcription factor capsulin controls spleen organogenesis. Proc Natl Acad Sci U S A . 2000; 97:9525–9530. doi: 10.1073/pnas.97.17.9525CrossrefMedlineGoogle Scholar
• 43. Kikuchi K, Gupta V, Wang J, Holdway JE, Wills AA, Fang Y, Poss KD. tcf21+ epicardial cells adopt non-myocardial fates during zebrafish heart development and regeneration. Development . 2011; 138:2895–2902. doi: 10.1242/dev.067041CrossrefMedlineGoogle Scholar
• 44. Katz TC, Singh MK, Degenhardt K, Rivera-Feliciano J, Johnson RL, Epstein JA, Tabin CJ. Distinct compartments of the proepicardial organ give rise to coronary vascular endothelial cells. Dev Cell . 2012; 22:639–650. doi: 10.1016/j.devcel.2012.01.012CrossrefMedlineGoogle Scholar
• 45. Levay AK, Peacock JD, Lu Y, Koch M, Hinton RB, Kadler KE, Lincoln J. Scleraxis is required for cell lineage differentiation and extracellular matrix remodeling during murine heart valve formation in vivo. Circ Res . 2008; 103:948–956. doi: 10.1161/CIRCRESAHA.108.177238LinkGoogle Scholar
• 46. Moore AW, McInnes L, Kreidberg J, Hastie ND, Schedl A. YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis. Development . 1999; 126:1845–1857.MedlineGoogle Scholar
• 47. Lu J, Richardson JA, Olson EN. Capsulin: a novel bHLH transcription factor expressed in epicardial progenitors and mesenchyme of visceral organs. Mech Dev . 1998; 73:23–32. doi: 10.1016/s0925-4773(98)00030-6CrossrefMedlineGoogle Scholar
• 48. Quaggin SE, Schwartz L, Cui S, Igarashi P, Deimling J, Post M, Rossant J. The basic-helix-loop-helix protein pod1 is critically important for kidney and lung organogenesis. Development . 1999; 126:5771–5783.MedlineGoogle Scholar
• 49. Christoffels VM, Grieskamp T, Norden J, Mommersteeg MT, Rudat C, Kispert A. Tbx18 and the fate of epicardial progenitors. Nature . 2009; 458:E8–9; discussion E9. doi: 10.1038/nature07916CrossrefMedlineGoogle Scholar
• 50. Christoffels VM, Mommersteeg MT, Trowe MO, Prall OW, de Gier-de Vries C, Soufan AT, Bussen M, Schuster-Gossler K, Harvey RP, Moorman AF, et al. . Formation of the venous pole of the heart from an Nkx2-5-negative precursor population requires Tbx18. Circ Res . 2006; 98:1555–1563. doi: 10.1161/01.RES.0000227571.84189.65LinkGoogle Scholar
• 51. Rinkevich Y, Mori T, Sahoo D, Xu PX, Bermingham JR, Weissman IL. Identification and prospective isolation of a mesothelial precursor lineage giving rise to smooth muscle cells and fibroblasts for mammalian internal organs, and their vasculature. Nat Cell Biol . 2012; 14:1251–1260. doi: 10.1038/ncb2610CrossrefMedlineGoogle Scholar
• 52. Guadix JA, Ruiz-Villalba A, Lettice L, Velecela V, Muñoz-Chápuli R, Hastie ND, Pérez-Pomares JM, Martínez-Estrada OM. Wt1 controls retinoic acid signalling in embryonic epicardium through transcriptional activation of Raldh2. Development . 2011; 138:1093–1097. doi: 10.1242/dev.044594CrossrefMedlineGoogle Scholar
• 53. Moss JB, Xavier-Neto J, Shapiro MD, Nayeem SM, McCaffery P, Dräger UC, Rosenthal N. Dynamic patterns of retinoic acid synthesis and response in the developing mammalian heart. Dev Biol . 1998; 199:55–71. doi: 10.1006/dbio.1998.8911CrossrefMedlineGoogle Scholar
• 54. Mahtab EA, Wijffels MC, Van Den Akker NM, Hahurij ND, Lie-Venema H, Wisse LJ, Deruiter MC, Uhrin P, Zaujec J, Binder BR, et al. . Cardiac malformations and myocardial abnormalities in podoplanin knockout mouse embryos: correlation with abnormal epicardial development. Dev Dyn . 2008; 237:847–857. doi: 10.1002/dvdy.21463CrossrefMedlineGoogle Scholar
• 55. Bochmann L, Sarathchandra P, Mori F, Lara-Pezzi E, Lazzaro D, Rosenthal N. Revealing new mouse epicardial cell markers through transcriptomics. PLoS One . 2010; 5:e11429. doi: 10.1371/journal.pone.0011429CrossrefMedlineGoogle Scholar
• 56. Vrancken Peeters MP, Mentink MM, Poelmann RE, Gittenberger-de Groot AC. Cytokeratins as a marker for epicardial formation in the quail embryo. Anat Embryol (Berl) . 1995; 191:503–508. doi: 10.1007/bf00186740MedlineGoogle Scholar
• 57. Rudat C, Kispert A. Wt1 and epicardial fate mapping. Circ Res . 2012; 111:165–169. doi: 10.1161/CIRCRESAHA.112.273946LinkGoogle Scholar
• 58. Wilm B, Ipenberg A, Hastie ND, Burch JB, Bader DM. The serosal mesothelium is a major source of smooth muscle cells of the gut vasculature. Development . 2005; 132:5317–5328. doi: 10.1242/dev.02141CrossrefMedlineGoogle Scholar
• 59. Asahina K, Zhou B, Pu WT, Tsukamoto H. Septum transversum-derived mesothelium gives rise to hepatic stellate cells and perivascular mesenchymal cells in developing mouse liver. Hepatology . 2011; 53:983–995. doi: 10.1002/hep.24119CrossrefMedlineGoogle Scholar
• 60. Dixit R, Ai X, Fine A. Derivation of lung mesenchymal lineages from the fetal mesothelium requires hedgehog signaling for mesothelial cell entry. Development . 2013; 140:4398–4406. doi: 10.1242/dev.098079CrossrefMedlineGoogle Scholar
• 61. Paris ND, Coles GL, Ackerman KG. Wt1 and β-catenin cooperatively regulate diaphragm development in the mouse. Dev Biol . 2015; 407:40–56. doi: 10.1016/j.ydbio.2015.08.009CrossrefMedlineGoogle Scholar
• 62. Chau YY, Bandiera R, Serrels A, Martínez-Estrada OM, Qing W, Lee M, Slight J, Thornburn A, Berry R, McHaffie S, et al. . Visceral and subcutaneous fat have different origins and evidence supports a mesothelial source. Nat Cell Biol . 2014; 16:367–375. doi: 10.1038/ncb2922CrossrefMedlineGoogle Scholar
• 63. Pryce BA, Brent AE, Murchison ND, Tabin CJ, Schweitzer R. Generation of transgenic tendon reporters, ScxGFP and ScxAP, using regulatory elements of the scleraxis gene. Dev Dyn . 2007; 236:1677–1682. doi: 10.1002/dvdy.21179CrossrefMedlineGoogle Scholar
• 64. Carmona R, González-Iriarte M, Pérez-Pomares JM, Muñoz-Chápuli R. Localization of the Wilm's tumour protein WT1 in avian embryos. Cell Tissue Res . 2001; 303:173–186. doi: 10.1007/s004410000307CrossrefMedlineGoogle Scholar
• 65. Martínez-Estrada OM, Lettice LA, Essafi A, Guadix JA, Slight J, Velecela V, Hall E, Reichmann J, Devenney PS, Hohenstein P, et al. . Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of snail and e-cadherin. Nat Genet . 2010; 42:89–93. doi: 10.1038/ng.494CrossrefMedlineGoogle Scholar
• 66. von Gise A, Zhou B, Honor LB, Ma Q, Petryk A, Pu WT. WT1 regulates epicardial epithelial to mesenchymal transition through β-catenin and retinoic acid signaling pathways. Dev Biol . 2011; 356:421–431. doi: 10.1016/j.ydbio.2011.05.668CrossrefMedlineGoogle Scholar
• 67. Velecela V, Lettice LA, Chau YY, Slight J, Berry RL, Thornburn A, Gunst QD, van den Hoff M, Reina M, Martínez FO, et al. . WT1 regulates the expression of inhibitory chemokines during heart development. Hum Mol Genet . 2013; 22:5083–5095. doi: 10.1093/hmg/ddt358CrossrefMedlineGoogle Scholar
• 68. Norden J, Grieskamp T, Lausch E, van Wijk B, van den Hoff MJ, Englert C, Petry M, Mommersteeg MT, Christoffels VM, Niederreither K, et al. . Wt1 and retinoic acid signaling in the subcoelomic mesenchyme control the development of the pleuropericardial membranes and the sinus horns. Circ Res . 2010; 106:1212–1220. doi: 10.1161/CIRCRESAHA.110.217455LinkGoogle Scholar
• 69. Wu M, Smith CL, Hall JA, Lee I, Luby-Phelps K, Tallquist MD. Epicardial spindle orientation controls cell entry into the myocardium. Dev Cell . 2010; 19:114–125. doi: 10.1016/j.devcel.2010.06.011CrossrefMedlineGoogle Scholar
• 70. Funato N, Ohyama K, Kuroda T, Nakamura M. Basic helix-loop-helix transcription factor epicardin/capsulin/Pod-1 suppresses differentiation by negative regulation of transcription. J Biol Chem . 2003; 278:7486–7493. doi: 10.1074/jbc.M212248200CrossrefMedlineGoogle Scholar
• 71. Piccolo S, Dupont S, Cordenonsi M. The biology of YAP/TAZ: hippo signaling and beyond. Physiol Rev . 2014; 94:1287–1312. doi: 10.1152/physrev.00005.2014CrossrefMedlineGoogle Scholar
• 72. Singh A, Ramesh S, Cibi DM, Yun LS, Li J, Li L, Manderfield LJ, Olson EN, Epstein JA, Singh MK. Hippo signaling mediators yap and taz are required in the epicardium for coronary vasculature development. Cell Rep . 2016; 15:1384–1393. doi: 10.1016/j.celrep.2016.04.027CrossrefMedlineGoogle Scholar
• 73. Xiao Y, Hill MC, Zhang M, Martin TJ, Morikawa Y, Wang S, Moise AR, Wythe JD, Martin JF. Hippo signaling plays an essential role in cell state transitions during cardiac fibroblast development. Dev Cell . 2018; 45:153–169.e6. doi: 10.1016/j.devcel.2018.03.019CrossrefMedlineGoogle Scholar
• 74. Landerholm TE, Dong XR, Lu J, Belaguli NS, Schwartz RJ, Majesky MW. A role for serum response factor in coronary smooth muscle differentiation from proepicardial cells. Development . 1999; 126:2053–2062.MedlineGoogle Scholar
• 75. Lu J, Landerholm TE, Wei JS, Dong XR, Wu SP, Liu X, Nagata K, Inagaki M, Majesky MW. Coronary smooth muscle differentiation from proepicardial cells requires rhoA-mediated actin reorganization and p160 rho-kinase activity. Dev Biol . 2001; 240:404–418. doi: 10.1006/dbio.2001.0403CrossrefMedlineGoogle Scholar
• 76. Small EM. The actin-MRTF-SRF gene regulatory axis and myofibroblast differentiation. J Cardiovasc Transl Res . 2012; 5:794–804. doi: 10.1007/s12265-012-9397-0CrossrefMedlineGoogle Scholar
• 77. Nelson TJ, Duncan SA, Misra RP. Conserved enhancer in the serum response factor promoter controls expression during early coronary vasculogenesis. Circ Res . 2004; 94:1059–1066. doi: 10.1161/01.RES.0000125296.14014.17LinkGoogle Scholar
• 78. Pipes GC, Creemers EE, Olson EN. The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis. Genes Dev . 2006; 20:1545–1556. doi: 10.1101/gad.1428006CrossrefMedlineGoogle Scholar
• 79. Olson EN, Nordheim A. Linking actin dynamics and gene transcription to drive cellular motile functions. Nat Rev Mol Cell Biol . 2010; 11:353–365. doi: 10.1038/nrm2890CrossrefMedlineGoogle Scholar
• 80. Braitsch CM, Combs MD, Quaggin SE, Yutzey KE. Pod1/Tcf21 is regulated by retinoic acid signaling and inhibits differentiation of epicardium-derived cells into smooth muscle in the developing heart. Dev Biol . 2012; 368:345–357. doi: 10.1016/j.ydbio.2012.06.002CrossrefMedlineGoogle Scholar
• 81. Xu J, Lamouille S, Derynck R. TGF-beta-induced epithelial to mesenchymal transition. Cell Res . 2009; 19:156–172. doi: 10.1038/cr.2009.5CrossrefMedlineGoogle Scholar
• 82. Molin DG, Bartram U, Van der Heiden K, Van Iperen L, Speer CP, Hierck BP, Poelmann RE, Gittenberger-de-Groot AC. Expression patterns of Tgfbeta1-3 associate with myocardialisation of the outflow tract and the development of the epicardium and the fibrous heart skeleton. Dev Dyn . 2003; 227:431–444. doi: 10.1002/dvdy.10314CrossrefMedlineGoogle Scholar
• 83. Sridurongrit S, Larsson J, Schwartz R, Ruiz-Lozano P, Kaartinen V. Signaling via the Tgf-beta type I receptor Alk5 in heart development. Dev Biol . 2008; 322:208–218. doi: 10.1016/j.ydbio.2008.07.038CrossrefMedlineGoogle Scholar
• 84. Hill CR, Sanchez NS, Love JD, Arrieta JA, Hong CC, Brown CB, Austin AF, Barnett JV. BMP2 signals loss of epithelial character in epicardial cells but requires the Type III TGFβ receptor to promote invasion. Cell Signal . 2012; 24:1012–1022. doi: 10.1016/j.cellsig.2011.12.022CrossrefMedlineGoogle Scholar
• 85. Clark CR, Robinson JY, Sanchez NS, Townsend TA, Arrieta JA, Merryman WD, Trykall DZ, Olivey HE, Hong CC, Barnett JV. Common pathways regulate type III TGFβ receptor-dependent cell invasion in epicardial and endocardial cells. Cell Signal . 2016; 28:688–698. doi: 10.1016/j.cellsig.2016.03.004CrossrefMedlineGoogle Scholar
• 86. Compton LA, Potash DA, Mundell NA, Barnett JV. Transforming growth factor-beta induces loss of epithelial character and smooth muscle cell differentiation in epicardial cells. Dev Dyn . 2006; 235:82–93. doi: 10.1002/dvdy.20629CrossrefMedlineGoogle Scholar
• 87. Trembley MA, Velasquez LS, Small EM. Epicardial outgrowth culture assay and ex vivo assessment of epicardial-derived cell migration. J Vis Exp . 2016. doi: 10.3791/53750CrossrefMedlineGoogle Scholar
• 88. Craig EA, Austin AF, Vaillancourt RR, Barnett JV, Camenisch TD. TGFβ2-mediated production of hyaluronan is important for the induction of epicardial cell differentiation and invasion. Exp Cell Res . 2010; 316:3397–3405. doi: 10.1016/j.yexcr.2010.07.006CrossrefMedlineGoogle Scholar
• 89. Bloomekatz J, Singh R, Prall OW, Dunn AC, Vaughan M, Loo CS, Harvey RP, Yelon D. Platelet-derived growth factor (PDGF) signaling directs cardiomyocyte movement toward the midline during heart tube assembly. Elife . 2017; 6:e21172. doi: 10.7554/eLife.21172CrossrefMedlineGoogle Scholar
• 90. Tallquist MD, Soriano P. Cell autonomous requirement for PDGFRalpha in populations of cranial and cardiac neural crest cells. Development . 2003; 130:507–518. doi: 10.1242/dev.00241CrossrefMedlineGoogle Scholar
• 91. Van Den Akker NM, Lie-Venema H, Maas S, Eralp I, DeRuiter MC, Poelmann RE, Gittenberger-De Groot AC. Platelet-derived growth factors in the developing avian heart and maturating coronary vasculature. Dev Dyn . 2005; 233:1579–1588. doi: 10.1002/dvdy.20476CrossrefMedlineGoogle Scholar
• 92. Smith CL, Baek ST, Sung CY, Tallquist MD. Epicardial-derived cell epithelial-to-mesenchymal transition and fate specification require PDGF receptor signaling. Circ Res . 2011; 108:e15–e26. doi: 10.1161/CIRCRESAHA.110.235531LinkGoogle Scholar
• 93. Mellgren AM, Smith CL, Olsen GS, Eskiocak B, Zhou B, Kazi MN, Ruiz FR, Pu WT, Tallquist MD. Platelet-derived growth factor receptor beta signaling is required for efficient epicardial cell migration and development of two distinct coronary vascular smooth muscle cell populations. Circ Res . 2008; 103:1393–1401. doi: 10.1161/CIRCRESAHA.108.176768LinkGoogle Scholar
• 94. Bax NA, Bleyl SB, Gallini R, Wisse LJ, Hunter J, Van Oorschot AA, Mahtab EA, Lie-Venema H, Goumans MJ, Betsholtz C, et al. . Cardiac malformations in Pdgfralpha mutant embryos are associated with increased expression of WT1 and Nkx2.5 in the second heart field. Dev Dyn . 2010; 239:2307–2317. doi: 10.1002/dvdy.22363CrossrefMedlineGoogle Scholar
• 95. Lin SL, Kisseleva T, Brenner DA, Duffield JS. Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney. Am J Pathol . 2008; 173:1617–1627. doi: 10.2353/ajpath.2008.080433CrossrefMedlineGoogle Scholar
• 96. Hamilton TG, Klinghoffer RA, Corrin PD, Soriano P. Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms. Mol Cell Biol . 2003; 23:4013–4025. doi: 10.1128/mcb.23.11.4013-4025.2003CrossrefMedlineGoogle Scholar
• 97. Lighthouse JK, Small EM. Transcriptional control of cardiac fibroblast plasticity. J Mol Cell Cardiol . 2016; 91:52–60. doi: 10.1016/j.yjmcc.2015.12.016CrossrefMedlineGoogle Scholar
• 98. Eralp I, Lie-Venema H, Bax NA, Wijffels MC, Van Der Laarse A, Deruiter MC, Bogers AJ, Van Den Akker NM, Gourdie RG, Schalij MJ, et al. . Epicardium-derived cells are important for correct development of the Purkinje fibers in the avian heart. Anat Rec A Discov Mol Cell Evol Biol . 2006; 288:1272–1280. doi: 10.1002/ar.a.20398CrossrefMedlineGoogle Scholar
• 99. Gittenberger-de Groot AC, Vrancken Peeters MP, Mentink MM, Gourdie RG, Poelmann RE. Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions. Cir Res . 1998; 82:1043–1052. doi: 10.1161/01.res.82.10.1043LinkGoogle Scholar
• 100. Miyagishi M, Hatta M, Ohshima T, Ishida J, Fujii R, Nakajima T, Fukamizu A. Cell type-dependent transactivation or repression of mesoderm-restricted basic helix-loop-helix protein, POD-1/Capsulin. Mol Cell Biochem . 2000; 205:141–147. doi: 10.1023/a:1007057611868CrossrefMedlineGoogle Scholar
• 101. Stennard FA, Harvey RP. T-box transcription factors and their roles in regulatory hierarchies in the developing heart. Development . 2005; 132:4897–4910. doi: 10.1242/dev.02099CrossrefMedlineGoogle Scholar
• 102. Wu SP, Dong XR, Regan JN, Su C, Majesky MW. Tbx18 regulates development of the epicardium and coronary vessels. Dev Biol . 2013; 383:307–320. doi: 10.1016/j.ydbio.2013.08.019CrossrefMedlineGoogle Scholar
• 103. Greulich F, Farin HF, Schuster-Gossler K, Kispert A. Tbx18 function in epicardial development. Cardiovasc Res . 2012; 96:476–483. doi: 10.1093/cvr/cvs277CrossrefMedlineGoogle Scholar
• 104. Grieskamp T, Rudat C, Lüdtke TH, Norden J, Kispert A. Notch signaling regulates smooth muscle differentiation of epicardium-derived cells. Circ Res . 2011; 108:813–823. doi: 10.1161/CIRCRESAHA.110.228809LinkGoogle Scholar
• 105. Red-Horse K, Ueno H, Weissman IL, Krasnow MA. Coronary arteries form by developmental reprogramming of venous cells. Nature . 2010; 464:549–553. doi: 10.1038/nature08873CrossrefMedlineGoogle Scholar
• 106. Tian X, Pu WT, Zhou B. Cellular origin and developmental program of coronary angiogenesis. Circ Res . 2015; 116:515–530. doi: 10.1161/CIRCRESAHA.116.305097LinkGoogle Scholar
• 107. Lavine KJ, Yu K, White AC, Zhang X, Smith C, Partanen J, Ornitz DM. Endocardial and epicardial derived FGF signals regulate myocardial proliferation and differentiation in vivo. Dev Cell . 2005; 8:85–95. doi: 10.1016/j.devcel.2004.12.002CrossrefMedlineGoogle Scholar
• 108. Pennisi DJ, Mikawa T. Normal patterning of the coronary capillary plexus is dependent on the correct transmural gradient of FGF expression in the myocardium. Dev Biol . 2005; 279:378–390. doi: 10.1016/j.ydbio.2004.12.028CrossrefMedlineGoogle Scholar
• 109. Vega-Hernández M, Kovacs A, De Langhe S, Ornitz DM. FGF10/FGFR2b signaling is essential for cardiac fibroblast development and growth of the myocardium. Development . 2011; 138:3331–3340. doi: 10.1242/dev.064410CrossrefMedlineGoogle Scholar
• 110. Shen H, Cavallero S, Estrada KD, Sandovici I, Kumar SR, Makita T, Lien CL, Constancia M, Sucov HM. Extracardiac control of embryonic cardiomyocyte proliferation and ventricular wall expansion. Cardiovasc Res . 2015; 105:271–278. doi: 10.1093/cvr/cvu269CrossrefMedlineGoogle Scholar
• 111. Li P, Cavallero S, Gu Y, Chen TH, Hughes J, Hassan AB, Brüning JC, Pashmforoush M, Sucov HM. IGF signaling directs ventricular cardiomyocyte proliferation during embryonic heart development. Development . 2011; 138:1795–1805. doi: 10.1242/dev.054338CrossrefMedlineGoogle Scholar
• 112. Wang K, Shen H, Gan P, Cavallero S, Kumar SR, Lien CL, Sucov HM. Differential roles of insulin like growth factor 1 receptor and insulin receptor during embryonic heart development. BMC Dev Biol . 2019; 19:5. doi: 10.1186/s12861-019-0186-8CrossrefMedlineGoogle Scholar
• 113. Kastner P, Grondona JM, Mark M, Gansmuller A, LeMeur M, Decimo D, Vonesch JL, Dollé P, Chambon P. Genetic analysis of RXR alpha developmental function: convergence of RXR and RAR signaling pathways in heart and eye morphogenesis. Cell . 1994; 78:987–1003. doi: 10.1016/0092-8674(94)90274-7CrossrefMedlineGoogle Scholar
• 114. Sucov HM, Dyson E, Gumeringer CL, Price J, Chien KR, Evans RM. RXR alpha mutant mice establish a genetic basis for vitamin A signaling in heart morphogenesis. Genes Dev . 1994; 8:1007–1018. doi: 10.1101/gad.8.9.1007CrossrefMedlineGoogle Scholar
• 115. Cunningham TJ, Duester G. Mechanisms of retinoic acid signalling and its roles in organ and limb development. Nat Rev Mol Cell Biol . 2015; 16:110–123. doi: 10.1038/nrm3932CrossrefMedlineGoogle Scholar
• 116. Niederreither K, Subbarayan V, Dollé P, Chambon P. Embryonic retinoic acid synthesis is essential for early mouse post-implantation development. Nat Genet . 1999; 21:444–448. doi: 10.1038/7788CrossrefMedlineGoogle Scholar
• 117. Stevens SM, von Gise A, VanDusen N, Zhou B, Pu WT. Epicardium is required for cardiac seeding by yolk sac macrophages, precursors of resident macrophages of the adult heart. Dev Biol . 2016; 413:153–159. doi: 10.1016/j.ydbio.2016.03.014CrossrefMedlineGoogle Scholar
• 118. Männer J, Schlueter J, Brand T. Experimental analyses of the function of the proepicardium using a new microsurgical procedure to induce loss-of-proepicardial-function in chick embryos. Dev Dyn . 2005; 233:1454–1463. doi: 10.1002/dvdy.20487CrossrefMedlineGoogle Scholar
• 119. Hellström M, Kalén M, Lindahl P, Abramsson A, Betsholtz C. Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development . 1999; 126:3047–3055.CrossrefMedlineGoogle Scholar
• 120. Harrison MR, Bussmann J, Huang Y, Zhao L, Osorio A, Burns CG, Burns CE, Sucov HM, Siekmann AF, Lien CL. Chemokine-guided angiogenesis directs coronary vasculature formation in zebrafish. Dev Cell . 2015; 33:442–454. doi: 10.1016/j.devcel.2015.04.001CrossrefMedlineGoogle Scholar
• 121. Cavallero S, Shen H, Yi C, Lien CL, Kumar SR, Sucov HM. CXCL12 signaling is essential for maturation of the ventricular coronary endothelial plexus and establishment of functional coronary circulation. Dev Cell . 2015; 33:469–477. doi: 10.1016/j.devcel.2015.03.018CrossrefMedlineGoogle Scholar
• 122. Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabé-Heider F, Walsh S, Zupicich J, Alkass K, Buchholz BA, Druid H, et al. . Evidence for cardiomyocyte renewal in humans. Science . 2009; 324:98–102. doi: 10.1126/science.1164680CrossrefMedlineGoogle Scholar
• 123. Uygur A, Lee RT. Mechanisms of cardiac regeneration. Dev Cell . 2016; 36:362–374. doi: 10.1016/j.devcel.2016.01.018CrossrefMedlineGoogle Scholar
• 124. van Wijk B, Gunst QD, Moorman AF, van den Hoff MJ. Cardiac regeneration from activated epicardium. PLoS One . 2012; 7:e44692. doi: 10.1371/journal.pone.0044692CrossrefMedlineGoogle Scholar
• 125. Pinto AR, Ilinykh A, Ivey MJ, Kuwabara JT, D'Antoni ML, Debuque R, Chandran A, Wang L, Arora K, Rosenthal NA, et al. . Revisiting cardiac cellular composition. Circ Res . 2016; 118:400–409. doi: 10.1161/CIRCRESAHA.115.307778LinkGoogle Scholar
• 126. Fu X, Khalil H, Kanisicak O, Boyer JG, Vagnozzi RJ, Maliken BD, Sargent MA, Prasad V, Valiente-Alandi I, Blaxall BC, et al. . Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart. J Clin Invest . 2018; 128:2127–2143. doi: 10.1172/JCI98215CrossrefMedlineGoogle Scholar
• 127. Valiente-Alandi I, Potter SJ, Salvador AM, Schafer AE, Schips T, Carrillo-Salinas F, Gibson AM, Nieman ML, Perkins C, Sargent MA, et al. . Inhibiting fibronectin attenuates fibrosis and improves cardiac function in a model of heart failure. Circulation . 2018; 138:1236–1252. doi: 10.1161/CIRCULATIONAHA.118.034609LinkGoogle Scholar
• 128. Xiang FL, Fang M, Yutzey KE. Loss of β-catenin in resident cardiac fibroblasts attenuates fibrosis induced by pressure overload in mice. Nat Commun . 2017; 8:712. doi: 10.1038/s41467-017-00840-wCrossrefMedlineGoogle Scholar
• 129. Molkentin JD, Bugg D, Ghearing N, Dorn LE, Kim P, Sargent MA, Gunaje J, Otsu K, Davis J. Fibroblast-specific genetic manipulation of p38 mitogen-activated protein kinase in vivo reveals its central regulatory role in fibrosis. Circulation . 2017; 136:549–561. doi: 10.1161/CIRCULATIONAHA.116.026238LinkGoogle Scholar
• 130. Braitsch CM, Kanisicak O, van Berlo JH, Molkentin JD, Yutzey KE. Differential expression of embryonic epicardial progenitor markers and localization of cardiac fibrosis in adult ischemic injury and hypertensive heart disease. J Mol Cell Cardiol . 2013; 65:108–119. doi: 10.1016/j.yjmcc.2013.10.005CrossrefMedlineGoogle Scholar
• 131. Quijada P, Misra A, Velasquez LS, Burke RM, Lighthouse JK, Mickelsen DM, Dirkx RA, Small EM. Pre-existing fibroblasts of epicardial origin are the primary source of pathological fibrosis in cardiac ischemia and aging. J Mol Cell Cardiol . 2019; 129:92–104. doi: 10.1016/j.yjmcc.2019.01.015CrossrefMedlineGoogle Scholar
• 132. Liu J, Wang H, Li J. Inflammation and inflammatory cells in myocardial infarction and reperfusion injury: a double-edged sword. Clin Med Insights Cardiol . 2016; 10:79–84. doi: 10.4137/CMC.S33164CrossrefMedlineGoogle Scholar
• 133. Huang GN, Thatcher JE, McAnally J, Kong Y, Qi X, Tan W, DiMaio JM, Amatruda JF, Gerard RD, Hill JA, et al. . C/EBP transcription factors mediate epicardial activation during heart development and injury. Science . 2012; 338:1599–1603. doi: 10.1126/science.1229765CrossrefMedlineGoogle Scholar
• 134. Balmer GM, Bollini S, Dubé KN, Martinez-Barbera JP, Williams O, Riley PR. Dynamic haematopoietic cell contribution to the developing and adult epicardium. Nat Commun . 2014; 5:4054. doi: 10.1038/ncomms5054CrossrefMedlineGoogle Scholar
• 135. Pinto AR, Godwin JW, Chandran A, Hersey L, Ilinykh A, Debuque R, Wang L, Rosenthal NA. Age-related changes in tissue macrophages precede cardiac functional impairment. Aging (Albany NY) . 2014; 6:399–413. doi: 10.18632/aging.100669CrossrefMedlineGoogle Scholar
• 136. Sanz-Morejón A, García-Redondo AB, Reuter H, Marques IJ, Bates T, Galardi-Castilla M, Große A, Manig S, Langa X, Ernst A, et al. . Wilms tumor 1b expression defines a pro-regenerative macrophage subtype and is required for organ regeneration in the zebrafish. Cell Rep . 2019; 28:1296–1306.e6. doi: 10.1016/j.celrep.2019.06.091CrossrefMedlineGoogle Scholar
• 137. Ramjee V, Li D, Manderfield LJ, Liu F, Engleka KA, Aghajanian H, Rodell CB, Lu W, Ho V, Wang T, et al. . Epicardial YAP/TAZ orchestrate an immunosuppressive response following myocardial infarction. J Clin Invest . 2017; 127:899–911. doi: 10.1172/JCI88759CrossrefMedlineGoogle Scholar
• 138. Patel VB, Shah S, Verma S, Oudit GY. Epicardial adipose tissue as a metabolic transducer: role in heart failure and coronary artery disease. Heart Fail Rev . 2017; 22:889–902. doi: 10.1007/s10741-017-9644-1CrossrefMedlineGoogle Scholar
• 139. Yamaguchi Y, Cavallero S, Patterson M, Shen H, Xu J, Kumar SR, Sucov HM. Adipogenesis and epicardial adipose tissue: a novel fate of the epicardium induced by mesenchymal transformation and PPARγ activation. Proc Natl Acad Sci U S A . 2015; 112:2070–2075. doi: 10.1073/pnas.1417232112CrossrefMedlineGoogle Scholar
• 140. Agullo-Pascual E, Cerrone M, Delmar M. Arrhythmogenic cardiomyopathy and Brugada syndrome: diseases of the connexome. FEBS Lett . 2014; 588:1322–1330. doi: 10.1016/j.febslet.2014.02.008CrossrefMedlineGoogle Scholar
• 141. Delmar M, McKenna WJ. The cardiac desmosome and arrhythmogenic cardiomyopathies: from gene to disease. Circ Res . 2010; 107:700–714. doi: 10.1161/CIRCRESAHA.110.223412LinkGoogle Scholar
• 142. Iyer VR, Chin AJ. Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D). Am J Med Genet C Semin Med Genet . 2013; 163C:185–197. doi: 10.1002/ajmg.c.31368CrossrefMedlineGoogle Scholar
• 143. Corrado D, Basso C, Judge DP. Arrhythmogenic cardiomyopathy. Circ Res . 2017; 121:784–802. doi: 10.1161/CIRCRESAHA.117.309345LinkGoogle Scholar
• 144. Matthes SA, Taffet S, Delmar M. Plakophilin-2 and the migration, differentiation and transformation of cells derived from the epicardium of neonatal rat hearts. Cell Commun Adhes . 2011; 18:73–84. doi: 10.3109/15419061.2011.621561CrossrefMedlineGoogle Scholar
• 145. Lien CL, Harrison MR, Tuan TL, Starnes VA. Heart repair and regeneration: recent insights from zebrafish studies. Wound Repair Regen . 2012; 20:638–646. doi: 10.1111/j.1524-475X.2012.00814.xCrossrefMedlineGoogle Scholar
• 146. Sánchez-Iranzo H, Galardi-Castilla M, Sanz-Morejón A, González-Rosa JM, Costa R, Ernst A, Sainz de Aja J, Langa X, Mercader N. Transient fibrosis resolves via fibroblast inactivation in the regenerating zebrafish heart. Proc Natl Acad Sci U S A . 2018; 115:4188–4193. doi: 10.1073/pnas.1716713115CrossrefMedlineGoogle Scholar
• 147. Porrello ER, Mahmoud AI, Simpson E, Hill JA, Richardson JA, Olson EN, Sadek HA. Transient regenerative potential of the neonatal mouse heart. Science . 2011; 331:1078–1080. doi: 10.1126/science.1200708CrossrefMedlineGoogle Scholar
• 148. Lam NT, Sadek HA. Neonatal heart regeneration. Circulation . 2018; 138:412–423. doi: 10.1161/CIRCULATIONAHA.118.033648LinkGoogle Scholar
• 149. Wang J, Cao J, Dickson AL, Poss KD. Epicardial regeneration is guided by cardiac outflow tract and Hedgehog signalling. Nature . 2015; 522:226–230. doi: 10.1038/nature14325CrossrefMedlineGoogle Scholar
• 150. Wang J, Karra R, Dickson AL, Poss KD. Fibronectin is deposited by injury-activated epicardial cells and is necessary for zebrafish heart regeneration. Dev Biol . 2013; 382:427–435. doi: 10.1016/j.ydbio.2013.08.012CrossrefMedlineGoogle Scholar
• 151. Missinato MA, Tobita K, Romano N, Carroll JA, Tsang M. Extracellular component hyaluronic acid and its receptor Hmmr are required for epicardial EMT during heart regeneration. Cardiovasc Res . 2015; 107:487–498. doi: 10.1093/cvr/cvv190CrossrefMedlineGoogle Scholar
• 152. Karra R, Foglia MJ, Choi WY, Belliveau C, DeBenedittis P, Poss KD. Vegfaa instructs cardiac muscle hyperplasia in adult zebrafish. Proc Natl Acad Sci U S A . 2018; 115:8805–8810. doi: 10.1073/pnas.1722594115CrossrefMedlineGoogle Scholar
• 153. Lepilina A, Coon AN, Kikuchi K, Holdway JE, Roberts RW, Burns CG, Poss KD. A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell . 2006; 127:607–619. doi: 10.1016/j.cell.2006.08.052CrossrefMedlineGoogle Scholar
• 154. Huang Y, Harrison MR, Osorio A, Kim J, Baugh A, Duan C, Sucov HM, Lien CL. Igf signaling is required for cardiomyocyte proliferation during zebrafish heart development and regeneration. PLoS One . 2013; 8:e67266. doi: 10.1371/journal.pone.0067266CrossrefMedlineGoogle Scholar
• 155. Kim J, Wu Q, Zhang Y, Wiens KM, Huang Y, Rubin N, Shimada H, Handin RI, Chao MY, Tuan TL, et al. . PDGF signaling is required for epicardial function and blood vessel formation in regenerating zebrafish hearts. Proc Natl Acad Sci U S A . 2010; 107:17206–17210. doi: 10.1073/pnas.0915016107CrossrefMedlineGoogle Scholar
• 156. Lien CL, Schebesta M, Makino S, Weber GJ, Keating MT. Gene expression analysis of zebrafish heart regeneration. PLoS Biol . 2006; 4:e260. doi: 10.1371/journal.pbio.0040260CrossrefMedlineGoogle Scholar
• 157. Wills AA, Holdway JE, Major RJ, Poss KD. Regulated addition of new myocardial and epicardial cells fosters homeostatic cardiac growth and maintenance in adult zebrafish. Development . 2008; 135:183–192. doi: 10.1242/dev.010363CrossrefMedlineGoogle Scholar
• 158. Zhao L, Borikova AL, Ben-Yair R, Guner-Ataman B, MacRae CA, Lee RT, Burns CG, Burns CE. Notch signaling regulates cardiomyocyte proliferation during zebrafish heart regeneration. Proc Natl Acad Sci U S A . 2014; 111:1403–1408. doi: 10.1073/pnas.1311705111CrossrefMedlineGoogle Scholar
• 159. Lowe V, Wisniewski L, Sayers J, Evans I, Frankel P, Mercader-Huber N, Zachary IC, Pellet-Many C. Neuropilin 1 mediates epicardial activation and revascularization in the regenerating zebrafish heart. Development . 2019; 146:dev174482. doi: 10.1242/dev.174482CrossrefMedlineGoogle Scholar
• 160. Cao J, Wang J, Jackman CP, Cox AH, Trembley MA, Balowski JJ, Cox BD, De Simone A, Dickson AL, Di Talia S, et al. . Tension creates an endoreplication wavefront that leads regeneration of epicardial tissue. Dev Cell . 2017; 42:600–615.e4. doi: 10.1016/j.devcel.2017.08.024CrossrefMedlineGoogle Scholar
• 161. Mizutani M, Wu JC, Nusse R. Fibrosis of the neonatal mouse heart after cryoinjury is accompanied by Wnt signaling activation and epicardial-to-mesenchymal transition. J Am Heart Assoc . 2016; 5:e002457. doi: 10.1161/JAHA.115.002457LinkGoogle Scholar
• 162. Cai W, Tan J, Yan J, Zhang L, Cai X, Wang H, Liu F, Ye M, Cai CL. Limited regeneration potential with minimal epicardial progenitor conversions in the neonatal mouse heart after injury. Cell Rep . 2019; 28:190–201.e3. doi: 10.1016/j.celrep.2019.06.003CrossrefMedlineGoogle Scholar
• 163. Rubin N, Darehzereshki A, Bellusci S, Kaartinen V, Ling Lien C. FGF10 signaling enhances epicardial cell expansion during neonatal mouse heart repair. J Cardiovasc Dis Diagn . 2013; 1:101. doi: 10.4172/2329-9517.1000101MedlineGoogle Scholar
• 164. Smart N, Bollini S, Dubé KN, Vieira JM, Zhou B, Davidson S, Yellon D, Riegler J, Price AN, Lythgoe MF, et al. . De novo cardiomyocytes from within the activated adult heart after injury. Nature . 2011; 474:640–644. doi: 10.1038/nature10188CrossrefMedlineGoogle Scholar
• 165. Smart N, Risebro CA, Melville AA, Moses K, Schwartz RJ, Chien KR, Riley PR. Thymosin beta-4 is essential for coronary vessel development and promotes neovascularization via adult epicardium. Ann N Y Acad Sci . 2007; 1112:171–188. doi: 10.1196/annals.1415.000CrossrefMedlineGoogle Scholar
• 166. Rui L, Yu N, Hong L, Feng H, Chunyong H, Jian M, Zhe Z, Shengshou H. Extending the time window of mammalian heart regeneration by thymosin beta 4. J Cell Mol Med . 2014; 18:2417–2424. doi: 10.1111/jcmm.12421CrossrefMedlineGoogle Scholar
• 167. Das S, Goldstone AB, Wang H, Farry J, D'Amato G, Paulsen MJ, Eskandari A, Hironaka CE, Phansalkar R, Sharma B, et al. . A unique collateral artery development program promotes neonatal heart regeneration. Cell . 2019; 176:1128–1142.e18. doi: 10.1016/j.cell.2018.12.023CrossrefMedlineGoogle Scholar
• 168. Aurora AB, Porrello ER, Tan W, Mahmoud AI, Hill JA, Bassel-Duby R, Sadek HA, Olson EN. Macrophages are required for neonatal heart regeneration. J Clin Invest . 2014; 124:1382–1392. doi: 10.1172/JCI72181CrossrefMedlineGoogle Scholar
• 169. Moerkamp AT, Lodder K, van Herwaarden T, Dronkers E, Dingenouts CK, Tengström FC, van Brakel TJ, Goumans MJ, Smits AM. Human fetal and adult epicardial-derived cells: a novel model to study their activation. Stem Cell Res Ther . 2016; 7:174. doi: 10.1186/s13287-016-0434-9CrossrefMedlineGoogle Scholar
• 170. Cao J, Poss KD. Explant culture of adult zebrafish hearts for epicardial regeneration studies. Nat Protoc . 2016; 11:872–881. doi: 10.1038/nprot.2016.049CrossrefMedlineGoogle Scholar
• 171. Witty AD, Mihic A, Tam RY, Fisher SA, Mikryukov A, Shoichet MS, Li RK, Kattman SJ, Keller G. Generation of the epicardial lineage from human pluripotent stem cells. Nat Biotechnol . 2014; 32:1026–1035. doi: 10.1038/nbt.3002CrossrefMedlineGoogle Scholar
• 172. Bargehr J, Ong LP, Colzani M, Davaapil H, Hofsteen P, Bhandari S, Gambardella L, Le Novère N, Iyer D, Sampaziotis F, et al. . Epicardial cells derived from human embryonic stem cells augment cardiomyocyte-driven heart regeneration. Nat Biotechnol . 2019; 37:895–906. doi: 10.1038/s41587-019-0197-9CrossrefMedlineGoogle Scholar
• 173. Wei K, Serpooshan V, Hurtado C, Diez-Cuñado M, Zhao M, Maruyama S, Zhu W, Fajardo G, Noseda M, Nakamura K, et al. . Epicardial FSTL1 reconstitution regenerates the adult mammalian heart. Nature . 2015; 525:479–485. doi: 10.1038/nature15372CrossrefMedlineGoogle Scholar
• 174. Zangi L, Lui KO, von Gise A, Ma Q, Ebina W, Ptaszek LM, Später D, Xu H, Tabebordbar M, Gorbatov R, et al. . Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Nat Biotechnol . 2013; 31:898–907. doi: 10.1038/nbt.2682CrossrefMedlineGoogle Scholar
• 175. Cao J, Navis A, Cox BD, Dickson AL, Gemberling M, Karra R, Bagnat M, Poss KD. Single epicardial cell transcriptome sequencing identifies caveolin 1 as an essential factor in zebrafish heart regeneration. Development . 2016; 143:232–243. doi: 10.1242/dev.130534CrossrefMedlineGoogle Scholar

The Epicardium in Cardiac Repair From the Stem Cell View

Source: https://www.ahajournals.org/doi/10.1161/CIRCRESAHA.119.315857

Share this post