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It is readily apparent that the process of heart development is an intricate one, in which cells derived from many embryonic sources coalesce and coordinate their behaviors and development, resulting in the mature heart. The behaviors and mechanisms of this process are complex, and still incompletely understood. However, it is readily apparent that communication between diverse cell types must be involved in this process. The signaling that emanates from epicardial and endocardial sources is the focus of this review.
Exquisite control and regulation is required for development of a properly functioning heart. As the heart progresses from mesoderm to a simple tube to a complex four-chambered structure, each step along this progression requires precise regulation of cellular behavior by a variety of sources. The hypoblast [1,2], epiblast , endoderm , neural tissue [5-8], endocardium [9-13], and epicardium [9,10,14] all signal to developing/mature muscle or cells that are fated to become myocardium during development. Additionally, the developing/mature myocardium emits signals to several of these structures (as well as to itself) that are necessary for proper development and maintenance of the cardiovascular system [15-19]. The proper transmittal of these signals is dependent on cellular emission of signals, cellular response to these signals, and morphogenetic movements of the organ and embryo that manipulate these communicating cells into the proper position for this communication to occur. Our focus here is to review signaling from the epicardium and endocardium, focusing on events that occur during embryonic development.
While a thorough discussion of the early embryonic development of the components of the mature heart is not within the scope of this review, a brief review of the embryonic origins and mechanisms that give rise to these tissues is useful for this discussion. Numerous excellent and more exhaustive reviews of endocardial [20-24], epicardial [25-29], and myocardial [30-32] development and origins are available. To summarize, endocardial and myocardial precursors originate in lateral splanchnic mesoderm [33-35]. These cells coalesce to form the cardiac crescent, which then fuses to become the primitive heart tube . The rhythmically contracting primitive heart tube at this point consists of a tube of epithelial endocardium surrounded by an epithelial tube of myocardium, and a layer of cardiac jelly separates these two tubes. This tube displays polarity along the anterior–posterior axis, as gene products expressed at this stage demonstrate that some early cell fate decisions have been made [36-38]. The linear heart tube then begins the process of looping, which establishes the position of the future chambers of the heart. Through this process, the presumptive atria are positioned anterior to the portion of the heart tube that gives rise to the ventricles. During this process of looping, the myocardium induces cells of the endocardium to delaminate and migrate into the cardiac jelly that separates the epithelial myocardium and the epithelial endocardium [39,40]. These cells migrate into the cardiac cushions and undergo differentiation into components of the valves. Endocardial cushions develop in multiple locations during heart development and give rise to several septal/valvular structures , along with migratory neural crest that invade the heart and contribute to the aorticopulmonary septum . During the process of looping, the epithelial proepicardium migrates to the surface of the myocardium, proliferates rapidly, and covers the surface of the myocardium [42-44]. This epithelium persists through development and adulthood, comprising the epicardium and pericardium. A subpopulation of cells of the epicardium undergo an epithelial–mesenchymal transition, delaminate from the epithelial epicardium, and infiltrate the developing myocardium [42,45]. These mesenchymal cells give rise to the smooth muscle, vascular endothelium, and cardiac fibroblasts of the coronary vasculature .
The endocardium secretes signals that act upon other components of the developing heart in a paracrine manner, as well as factors that act upon the endocardium itself in an autocrine fashion. The endocardium also receives and responds to cues from other tissue sources during heart development, such as VEGF signaling from the myocardium that represses delamination of cells of the endocardial epithelium prior to endocardial cushion formation . These interactions lie outside the scope of this review. In this section, signals from the endocardium to the developing myocardium, cardiac mesenchyme, and endocardium will be discussed (Table 1).
During heart development, one of the first myocardial activities demonstrated to be affected by signaling from the endocardium is trabeculation of the myocardium. The EGF family member neuregulin-1 (NRG-1), which is expressed at a high level in the endocardium during development, has been demonstrated to act on both myocardium [13,46-50] and endocardial mesenchyme . Neuregulin-1 secreted from the endocardium stimulates heterodimerization of ErbB2 and Erb4, activating downstream signaling pathways that have been demonstrated to be critical for early cardiac trabeculation via both inactivation of neuregulin-1  and the ErbB2 receptor . NRG-1 also has recently been shown to be sufficient to induce differentiation of cardiomyocytes to components of the cardiac conduction system [13,49,50]. Through interaction with IGF-1, which is also secreted by the endocardium, NRG-1 controls cardiac chamber morphogenesis through activation of the phosphatidylinositiol 3-kinase . The synergistic effects of IGF-1 and neuregulin-1 also show themselves later in cardiac development, as myocardium treated with these two factors simultaneously displayed an increase in DNA synthesis and expansion of the compact zone and atrioventricular cushions later in development . However, NRG-1 not only acts on cells of the developing myocardium, it also triggers proliferation of endocardially derived mesenchyme during cardiac cushion development through stimulation of the ErbB2/ErbB3 receptor homodimer [51,52]. To summarize, neuregulin-1 signals from the endocardium control cellular behaviors of myocardium and endocardial mesenchyme, and affect the processes of differentiation and proliferation during cardiac development.
Work from the Markwald laboratory demonstrated that members of the FGF signaling pathway are expressed in the epicardium, endocardium, and myocardium during heart development . Mesenchymal derivatives of the endocardium express FGF-4 and FGFR1-FGFR3. This work indicated that endocardium and endocardially derived mesenchyme likely signaled in an autocrine fashion through the FGF-4 pathway, leading to downstream signal transduction through the Ras and PLC-γ pathways. In vivo experiments demonstrated that FGF-4 signaling induced proliferative expansion of the cushion mesenchyme during the formation of cardiac valve leaflets. The Ornitz group later showed that FGF-9, FGF-16, and FGF-20 are all expressed in the endocardium during mouse embryogenesis . These FGFs signal to the myocardium through FGFR1 and FGFR2, stimulating myocyte differentiation and proliferation.
Endothelins, a family of small signaling peptides, signal through their G-protein coupled receptors, ETA and ETB [54-57]. Two enzymes, endothelin-converting enzyme-1 and -2 (ECE-1 and ECE-2), are necessary for conversion of the biologically inactive precursor endothelins into biologically active products [58,59]. Endothelins are highly expressed in various endocardial domains, and receptors for ET family members are expressed in migratory and post-migratory neural crest derivatives of the heart and the myocardium itself . ECEs are expressed in the mesenchyme of endocardial cushions, mesenchyme of the brachial arch arteries, and in endocardial epithelia throughout the heart. Disruptions of endocardial endothelin signaling by inactivation of components of this pathway cause cardiac defects including defects in aortic arches, major arteries, and septum [57,60-62]. Interestingly, these septation defects involve the septation of the outflow tract and the septation of the common ventricle. Thus, endothelin signaling mediates septation by modulating behavior of neural crest derived cells of the OFT and endocardial/myocardial derived cells of the IVS.
Global inactivation of the neurofibromin (Nf1) gene in mice leads to gross cardiac anomalies that cause gestational lethality, including OFT defects, enlarged endocardial cushions, ventricular septal defects, and thinned myocardium [63-65]. The initial interpretation of this phenotype was that these defects were caused by disruption of Nf1 gene activity in neural crest cells and derivatives. However, work by the Epstein laboratory later demonstrated that endocardial specific inactivation of Nf1 gene activity was sufficient to generate a similar phenotype, indicating that endocardial Nf1 activity was more important than previously thought . Additionally, neural crest or myocardium specific inactivation of Nf1 did not yield cardiac defects. Although Nf1 is not secreted from the endocardium, Nf1 activity in the endocardium serves to modulate endocardial Ras GTPase and NFATc activity during heart development .
Stromal cell-derived factor 1, also called pre-B-cell growth-stimulating factor, is a member of the CXC family of chemokines . Along with a role in hematopoeisis, SDF-1 has a signaling role during development of the heart. Genetic inactivation of either the SDF-1 chemokine or the specific receptor for SDF-1, CXCR4, yield mice that die embryonically and display atrioventricular septum defects [68,69]. In situ analysis shows that SDF-1 is expressed in the endocardium of the mouse during cardiogenesis , while the CXCR4 receptor is expressed in the aorticopulmonary septum and the ventricular wall of the heart at this time [68,70]. This expression pattern indicates that endocardially derived SDF-1 signals are critical for septation of the heart.
As described here, it is clear that these endocardium-derived signals are critical for proper morphogenesis of the heart. There are undoubtedly other signals that originate from the endocardium that control developmental processes, both described and yet to be discovered. Additionally, myocardial development is also controlled by the epithelial lining on the outside of the heart muscle—the epicardium.
The proepicardium, epicardium, and epicardially derived cells are critical for normal heart development. These structures not only provide a major portion of the cells that comprise the coronary vasculature, the epicardium also plays an important role in modulating the development of the myocardium. Naturally, as diseases related to the coronary vasculature are a major cause of death, interest in understanding the normal development of the transient and definitive structures of this tissue has in recent years increased greatly. Interestingly, the mechanism by which the coronary vasculature is generated was long thought to be unique. However, recent work by Wilm and colleagues has demonstrated that a very similar mechanism generates components of the vasculature of the gut . Therefore, understanding of signaling mechanisms that play a role in cardiac vasculogenesis may well have implications for development and maintenance of tissues in other organs.
Early indications that the proper development of the myocardium was dependent upon close interaction and signaling from the myocardium were noted in studies involving inactivation of transcription factors and cell adhesion molecules. Knockouts of FOG-2 , WT-1 , VCAM-1 , and α4 integrin  all displayed cardiac defects, most notably abnormally thin myocardium. Some of these genes have expression patterns that are largely restricted to the epicardium, illustrating that disruption of normal epicardial function had deleterious effects on cardiac compaction and proliferation. Here, some of the specific factors that are involved in myocardial regulation by epicardium are discussed.
Cardiovascular developmental defects in experimental settings have long been associated with improper retinoic acid (RA) levels during gestation [76,77]. However, the molecular mechanisms underlying these observations are only recently becoming more apparent. Ingested vitamin A is converted to active retinoids by a series of oxidative reactions in vivo. Therefore, presence of components of this biosynthetic pathway are indicators of sources of RA signaling. One of these components, RALDH-2, is highly expressed in the developing epicardium of the mouse  and the chicken , but not in the myocardium. Global inactivation of one of the receptors for RA, RXR-α, caused embryonic death resulting from cardiovascular defects including myocardial thinning [80-82]. This seemingly indicates that RA–RXR-α signals from the epicardium to the myocardium may be critical for proper cardiogenesis. However, myocardium specific inactivation of RXR-α displayed no cardiac phenotype . Subsequent investigation utilizing an epicardial-specific inactivation of RXR-α yielded a similar phenotype to that of the global inactivation, pointing to the conclusion that an epicardial autocrine RA/ RXR-α signaling mechanism may regulate myocardial proliferation by inducing release of another trophic factor(s) from the epicardium , an idea which had previously been postulated [14,85]. Early investigations indicate that two of these factors may be FGF-2, which is discussed in greater detail below.
Mikawa and colleagues demonstrated that FGF-2 signaling from the epicardium was necessary for proliferation of myocytes in the outer compact myocardial wall , and further elucidation of the mechanism of the regulation of myocyte proliferation by epicardium has followed. However, previous studies had demonstrated only a mild phenotype when FGF-2 was inactivated [87,88]. Later studies demonstrated that FGF-9, -16, and -20 are all expressed in the epicardium, and that these growth factors signal to the myocardium through FGF receptors 1 and 2 . Knockout of these receptors in the myocardium demonstrate that this FGF signaling is critical for proper proliferation and differentiation of the cardiac muscle. These results correspond with those stated previously, namely that RXR-α inactivation in the epicardium led to a similar phenotype, as it has been shown that RA signaling to the epicardium induces expression of FGFs [14,83,85]. Subsequently, Ornitz and colleagues have revealed a role for epicardial FGF signaling for modulation of coronary vasculogenesis as well. This series of experiments shows that epicardial FGF signals induce myocardial activation of Hedgehog activation, that in turn upregulate VEGF-A, -B, -C, and angiopoietin-2 expression .
To summarize, evidence indicates that autocrine and paracrine RA signaling to the epicardium induces expression of FGF family members. The epicardium uses these growth factors to signal to the myocardium, thereby controlling myocyte proliferation, differentiation, and coronary vasculogenesis.
Thus, multiple signaling pathways originating from epithelia on either side of the myocardium regulate cardiac developmental processes. Migration, differentiation, and proliferation have all been demonstrated to be regulated by these extrinsic signals. The field’s increasing understanding of the developmental regulation exerted by these signaling pathways will lead to a greater understanding of human cardiovascular birth defects, which are one of the most commonly occurring birth defects. Further investigation into these signaling networks may also lead to a better understanding of how extra-myocardial signals support and control myocardial function in the adult organ.
The authors would like to thank members of our laboratory for helpful comments and discussion. DMB is supported by NIH HL37675 and NIH HL079050. TKS is supported by AHA 0415252B and NIH HL079050.