The Molecular and Cellular Mechanisms of Heart Pacemaker Development in Vertebrates

A small group of specialized myocardial cells constitutes a natural dominant heart pacemaker in the so-called sinoatrial node (SAN). The SAN determines proper heart automaticity throughout life in animals, including humans. The heart pacemaker is characterized by specific histological organization and unique pattern of gene expression, defining the electrophysiological phenotype of constituent cardiomyocytes. The SAN development starts very early in embryogenesis and continues until the late prenatal period. The clarification of the origin of the heart pacemaker and molecular mechanisms controlling its development promotes the elaboration of bio-artificial pacemakers and understanding of causes for many cardiovascular diseases, including hereditary, developmental or acquired arrhythmias. The investigation of SAN embryogenesis facilitates the solution of reprogramming problem for cardiomyocytes or somatic cells designed for cellular therapy of cardiovascular diseases and reversion of pathological heart remodeling. To date, significant progress has been achieved in the field of identifying genetic and molecular pathways that control the pacemaker cells' nature and govern morphological and functional maturation of the dominant cardiac pacemaker during ontogenesis. This review provides the information on the key transcription factors and molecular regulatory cascades (BMP, Wnt, Wt1, Slit/Robo, RhoA, podoplanin, VEGF, and PDGF) involved in (epi)genetic control of the pacemaker myocyte progenitors and determination of their electrophysiological phenotypes.

The heart is responsible for blood circulation in the organism throughout life starting from the earliest stages of prenatal ontogenesis. The heart's pumping function is provided by rhythmic coordinated sequence of excitation, contraction, and dilatation of chambers. Rhythmic heart excitation is governed by a small group of myocytes with a specific electrophysiological properties. The myogenic automaticity is typical for all vertebrates. These specialized cardiomyocytes are located in the right atrial wall between the entries of superior (right cranial) and inferior (posterior) venae cavae in mammals, thus constructing a dominant heart pacemaker called the sinoatrial node (SAN) according to its location.
The formation of the heart pacemaker during embryogenesis is crucial for its normal functioning and organism survival. Developmental disorders of pacemaker functioning are associated with multiple cardiovascular diseases. Many regulatory aspects of heart pacemaker development have already been discovered along with the establishment of a specific electrophysiological phenotype of cardiomyocytes. The present review focuses on the molecular mechanisms underlying the formation and establishment of pacemaker function during ontogenesis. It should be mentioned that the definitive SAN pacemaker is a sophisticated tissue structure consisting of specialized pacemaker cardiomyocytes, elements of connective tissue, and resident immune cells. Only the data regarding the ontogenesis of myocardial component of the heart pacemaker will be provided, although the rest of the cellular components, as the elements of microenvironment of cardiac myocytes, undoubtedly affect the development and function [1].
Cardiogenesis is a multistep process involving several structures producing progenitor cardiomyocytes and varying in the late stages of ontogenesis between different vertebrates. Therefore, the anatomy of a definitive heart pacemaker in vertebrates appears diverse and not fully understood in some cases. In particular, the pacemaker of bony fish is represented by a circular structure surrounding the sinoatrial valve (SAV); the elements of sinus venosus (SV) walls have pacemaker properties in amphibians. Nevertheless, the early stages of cardiogenesis and embryogenesis of the heart pacemaker are mainly similar in lower vertebrates or ectotherms, birds, and mammals [2]. The vast majority of data on transcription and molecular control of pacemaker development were obtained using chicken or mice embryos. It turned out that the mechanisms regulating the development of the heart pacemaker were conservative among amniotes and potentially among all the vertebrates, as determined by identical (epi)genetic programs and groups of regulatory factors. This is why the following experimental results can be extrapolated to all vertebrates and translated to SAN ontogenesis of humans [3,4].

Early Stages of Cardiogenesis
Elaboration of the problem of heart pacemaker formation is impossible without referring to the early embryonic cardiogenesis. All vertebrates' hearts have a mesodermal origin. Multipotent progenitor cells are determined at the early stages of embryogenesis. The induction and specification of cardiogenic cells occur at the stage of epiblast, or embryonic disk, formation in all amniotes and mammals, in particular. Cardiogenic cells as the progenitors of cardiomyocytes, as well as smooth muscle cells and endotheliocytes, migrate from two symmetrical bilateral epiblast segments through the walls of primary groove and Hensen's node to the space above forming endoderm becoming the cardiogenic part of mesoderm, while losing cell adhesion proteins, such as E-cadherins [5], under the influence of posterior body end gene products, e.g., Nodal, Vg1, etc. [6], signaling cascades activated by Wnt family proteins [7,8], BMP [9], and a number of other morphogenetic factors (FGF-1, 2, and 8) [10]. In humans bilaterally symmetrical cardiogenic parts of mesoderm fuse across the midline to form a crescent-shaped area, a cardiac crescent [11]. After the migration, cardiogenic cells remain in close contact with endoderm that produces cardiogenic factors.
During further development, cardiogenic cells are localized in the anterior part of the visceral layer of the lateral mesoderm. Soon after the formation of visceral cardiogenic mesoderm, the so-called heart fields can be identified in it-the first and second ones in accordance with the classical concepts. Mesodermal and mesenchymal cells deriving from bilateral parts of first heart field (FHF) form the primary heart tube typical for all vertebrate animals [12]. As a result of merging of two mesodermal germ layers, the heart tube is sur-rounded by the pericardial coelomic cavity. The elongation and thickening of heart tube walls and the formation of heart compartments are associated with the inclusion of migrating and differentiating mesenchymal cells from visceral mesoderm. The migration of mesodermal cells becomes possible due to their deepithelialization or the induction of so-called epithelialmesenchymal transition (EMT). The working myocardium of the left ventricle, part of the myocardium of the right and left atria of the mature heart, but not the pacemaker myocardium of SV and SAN are derivatives of the primary heart tube, i.e., are formed with mesoderm originating from FHF.

Automaticity of the Primary Cardiac Tube
The heart is the organ starting to function at the earliest stages of ontogenesis. Directly after the merging of mesodermal germ layers to form cardiac crescent in poorly differentiated cardiomyocytes, or cardioblasts, the sarcomere assembly, expression of membrane ion transporters and ion channels, particularly, proteins of sodium calcium exchanger and L-type calcium channels occur. Cardiogenic cells of mice demonstrate the ability for automaticity at stages Е7.75-Е8 by spontaneously rhythmically generating cytoplasmic Ca 2+ -waves and, thus, action potentials [13]. Later after the formation of heart tube the "primary" myocardium shows the ability to spontaneously contract when consisting of only two or three cell layers [14]. Spontaneous electrical activity is generated around the venous or posterior boundary, inducing the peristaltic contractions that determine the directed flow of blood from the inflow to outflow tract of the heart tube [15]. It is thought that susceptibility to the initial activation of the venous boundary of the heart tube is associated with the cranio-caudal gradient of concentrations of morphogenetic factors, particularly, of retinoic acid synthesized in posterior mesoderm, and the activity gradient of Wnt, BMP, and FGF protein families [16].
The automaticity of the heart tube was not related in any way to the activity of certain germs of the mature heart pacemaker (SAV and SAN). Currently, in contrast with the concepts developed in the 1990s, it has been found that the primary heart tube does not contain all elements of the mature heart, including the miniature germs of the heart pacemaker [17]. Myocytes of both the heart tube and SAN express hyperpolarization-activated cyclic nucleotide-gated (HCN) channels constituting I f pacemaker current [18,19]. Depolarizing I f ion current [20] mediated predominantly by HNC4 is one of the mechanisms providing the so-called slow diastolic depolarization potentiating spontaneous generation of action potentials in the pacemaker cells [21,22]. Cranio-caudal gradient of HCN4 expression can be also observed already at the stage of cardiac crescent before the formation of heart tube [19].
Kir2.x channels transduce inward rectifier potassium current that stabilizes the resting potential and prevent the abnormal activity in cardiomyocytes [23]. As aforementioned, promyocardial cells of heart tube construct the working myocardium of the left ventricle and atria as a result of their proliferation.
It is supposed that TBX3 and TBX4 transcriptional repressors interfere with the functioning of the Nkx2.5/TBX5/GATA4 complex up to a certain point of cardiogenesis, namely before the beginning of SV formation, while the expression of HCN4 is maintained by Isl1 [24]. It should be mentioned that HCNpositive cells of the heart tube also express Cx40 connexins typical for the working cardiomyocytes of atria and ventricles [20] but not for SAN.
To summarize, myocytes of the heart tube and SAN cardiomyocytes are characterized by different patterns of TF expression controlling the electrophysiological features. Cardiomyocytes of the primary heart tube are not involved in automaticity provided by the embryonic SV or mature SAN [25]. Progenitor SAN cells can be found in the embryonic heart much later than the moment of initiation of spontaneous heart tube contractions.

Second Heart Field as the Source of Procardiogenic Progenitor Cells of the Venous Pole of the Heart
The source of progenitor cardiomyocytes is the second heart field (SHF), which is one of the mesodermal segments [11,26,27]. Procardiogenic progenitor cells of SHF mesoderm are identified in epiblast of mammals. Mesodermal bilaterally symmetrical parts of SHF are located medially from the crescent of primary field [28]. The formation of cardiogenic mesenchymal cells from SHF mesoderm by means of EMT, together with their migration, proliferation [29], and differentiation to cardiomyocytes during ontogenesis, are happening substantially longer and later than in FHF. Mesenchymal cells expel from SHF after the migration of progenitor cells of the primary field has already been finished, they have been included into the wall of heart tube, and their differentiation has been directed towards the myocardial path. The formation of a new heart compartment is provided by the immigration of SHF cells and corresponds with the initiation of the heart tube looping phase. Several heart structures are constructed from the mesenchymal SHF progenitors, for example, SV, SAV, atrioventricular connection, right ventricle and interventricular septum, parts of atria, interatrial septum, and myocardial tissue in the walls of pulmonary veins and venae cavae [30,31].
The primary heart tube located in the coelomic cavity remains connected with the visceral mesoderm by so-called dorsal cardiogenic mesoderm, or dorsal mesocardium, represented by a longitudinal mesodermal band originating from transformed SHF. Later, the dorsal mesodermal band is partially separated from the heart tube, maintaining its connection only around arterial and venous poles and, thus, constituting the anterior and posterior regions of the dorsal mesoderm. In these two zones, cells are actively dividing and migrate to the arterial and venous poles of the heart tube to proliferate and differentiate in cardiomyocytes [32]. According to the standard model, the formation of venous and arterial poles of the primitive heart in vertebrates is provided by anterior and posterior heart fields, the regions of SHF [28,33]. Mesenchymal cells expelling from the posterior cardiogenic field begin to incorporate into the wall of the primary heart tube following the cells of the anterior field; therefore, the structures of the venous heart pole, including SV, develop later, starting from E9.5 in mice, than the arterial pole structures [17].
It was proposed that the posterior cardiogenic field was homogenous and its cells serve as the source of all venous pole structures, including embryonic SV and SAN [34]. Currently, it has been shown that visceral mesoderm before the stage of fields segmentation is more heterogeneous than has earlier been thought and Origin of specialized pacemaker cardiomyocytes of the sinoatrial node (SAN) from mesodermal heart fields during embryonic development and the expression pattern of crucial transcription factors (TFs: Mesp1, Nkx2.5, Isl1, TBX18, and TBX3) in mesodermal heart fields and cells derived from them at different stages of cardiogenesis in mammals. Mesenchymal progenitors, mature cardiomyocytes, and SAN fibroblasts likely originate from two pools (Nkx2.5 -/Isl1 -/TBX18 + and Isl1 + /Nkx2.5 -) of mesodermal cells. Yellow and red colors highlight the structures demonstrating the automatic or pacemaker activity at different stages of cardiogenesis. Expressed TF is shown in red script; the absence of TF expression is shown in dark script.

Specification
Procardiogenic mesoderm "First" heart field "First" heart field "Lateral rim" SV and SAN progenitors "Second" heart field

Molecular Markers of Mesoderm of Heart Fields and SAN
Currently, MesP1 is considered as the key factor of cardiogenic specification and mesoderm proliferation ( Fig. 1) [36]. Presumably, specifically the MesP1 controls a group of regulatory factors required for early cardiogenesis. FGF8, FGF10, Isl1, Id2, GATA4/6, Mef2c, podoplanin, and TF of TBX family are referred to MesP1-controlled TFs and regulatory proteins [37]. Such cells as FHF and SHF express MesP1 and TFs of GATA family [38].
Cardiogenic mesoderm divides into cell subpopulations prior to the formation of the heart tube, thus undergoing patterning by expressed TFs. FHF mesoderm sustains the expression of Isl1 and Nkx2.5; however, Isl1 expression in cardiomyocytes, which are first field descendants, is alleviated after the termination of primary heart tube formation. SHF mesoderm maintains the expression of only Isl1, although before the differentiation the cells of the second field restart the expression of Nkx2.5, whereas the Isl1 level is decreasing. Thus, the classification of cardiogenic fields is rather conditional and they can be considered as the mesoderm domains with complexly alternating TF profile during ontogenesis [35]. It is the TF expression pattern that is responsible for the specification of mesodermal cells as the progenitors of SAN pacemaker cardiomyocytes or working cardiomyocytes of atria and ventricles.
The origin of the heart pacemaker can be reconstructed by the determination of TF expression profile in cardiomyocytes of mature SAN and progenitor cells of cardiogenic mesoderm. It was found that a small cell subpopulation originating from the periphery of cardiac crescent mesoderm, i.e., lateral rim, loses Isl1 and Nkx2.5 expression before the entering to EMT and proliferation of mesenchyme but begins to express TBX18. Such Nkx2.5-and Isl-negative TBX18-positive (Nkx2.5 -/Isl1 -/TBX18 + ) cells are the progenitors of SV and probably of pacemakers in all vertebrate ani-mals. It has been demonstrated that the myocardium of SAN is built in mammals of cells derived from two different subpopulations: (1) Isl1-positive and Nkx2.5-negative (Isl1 + /Nkx2.5 -) cells of second, or posterior, heart field; (2) Nkx2.5 -/Isl1 -/TBX18 +cells of the lateral rim [35] (Fig. 1). It is significant that the cells of definitive SAN maintain the expression of Isl1, which are probably necessary for supporting the pacemaker phenotype and HCN4 expression. Besides, Nkx2.5 -/Isl1 -/TBX18 + cells are also supposed as precursors of epicardium, tissues of coronary vessels, and heart fibroblasts playing a significant role in SAN function.
Currently, it has been experimentally proven that specifically the part of mesechymal cells that express TBX18 migrate, proliferate, differentiate in cardiomyocytes of primordial SAN, and constitute the majority of mature SAN (around 75% of cells) [20] in mammals, whereas the removal of these cells at the early embryonic stages does not allow SAN to form.

Morphogenesis of Sinus Venosus
Sinus venosus (SV) is the first structure to be identified as an individual chamber during heart ontogenesis. SV is at first separated with the sinoatrial canal from the forming common atrium that is transformed into SAV in all vertebrates. SV originates as a result of myocardilization, i.e., immigration and incorporation of mesenchymal cells from SHF into the wall of proximal segments of common, right and left, cardinal veins, and/or the caudal part of the heart tube itself. As a result of myocardilization of cardinal veins, symmetrical elongated segments, the so-called SV horns, are formed. The horns comprising the most of VS in some animals, particularly, in mammals [41,42].
In mammals, SV is a temporary embryonic structure. During the prenatal period, SV is reduced so that its left horn transforms into coronary sinus draining coronary veins. The right horn of mammals is integrated into the wall of the right atrium during so called atrialization [24]. It is proposed that a part of SV myocardium can also be included into the wall of the left atrium. Anterior and posterior cardinal veins bringing blood to the SV at the embryonic stage are reorganized into superior caval (cranial) and azygous veins, respectively [43]. Therefore, SAN is a structure formed on the basis of the SV in all mammals.

Atrialization of Sinus Venosus in Mammals
SV atrialization provokes substantial reorganization of SAV consisting of two leaflets. SAV leaflets get distant and partially reduce during embryogenesis: the right SAV leaflet is included into the wall of the right atrium, dividing it into smooth walled and trabeculated portions, preserving as crista terminalis. A part of the right SAV leaflet also constructs the eustachian valve. The left SAV leaflet is included into the atrial septum from its right side. Finally, the wall of the right SV horn is modified into a part of the adult right atrial wall in mammals, which is located between the crista terminalis and atrial septum. The structure is also known as sinus venarum. SV incorporation appears to be incomplete in some mammals, which is why venae cavae entries open in the space partially separated from the right atrium [44].
In a number of studies, functional experiments discovered that the segment consisting of SV myocardium and cardinal veins plays the role of the heart pacemaker from the moment of termination of heart tube looping to the moment of complete SV atrialization during the prenatal development, rather than localized, well distinctive structure [43]. It should be noted that the myocardium of both cardinal veins and the entire SV expresses HCN4 channels until the late prenatal period [45,46]. Therefore, the tissue of SV and cardinal veins can function as embryonic pacemaker in different mammalian species outside the developing SAN [47,48].

Formation of Definitive Heart Pacemaker in Vertebrate Animals
No SV reduction occurs in lower vertebrates during the ontogenesis and its atrialization can be of a functional character only. Apparently, the role of the definitive pacemaker in amphibians is played by a whole SV myocardium derived from Nkx2.5 -/Isl1 -/TBX18 +cells with increased Isl1 expression by the end of embryogenesis. The functional atrialization of SV manifests in replacement of the majority of cardiomyocytes in SV by fibroblasts in other ectotherms, e.g., bony fish, or in lose of the ability for automaticity in reptiles. In this case, only the circular segment of myocardium on SAV periphery preserves the ability for automaticity [23].
Mesenchymal Isl1 + /Nkx2.5 --and Nkx2.5 -/Isl1 -/TBX18 + progenitors of SAN localize around the entries of right and/or left common cardinal veins, i.e., at the basis of SV horns, in mammals after the migration from heart fields. It is thought that the population of progenitor SAN cells is not mixed with the progenitors of SV itself, thus excluding its cellular mosaicism. In mice, primordial SAN develops as a thickened proximity of the right SV horn wall during E10.5-E14.5. In this zone, no Gja1 expression encoding Cx40 connexin occurs, unlike the expression of propacemaker TFs [49]. SV atrialization results in the incorporation of a part of myocardium at the proximity of cardinal veins containing primordial SAN into the wall of the right atrium and definitive SAN can be detected in the intercaval area.
The area of myocardium exhibiting automatic activity and including cardinal veins, SV, and SAN reduces in the course of late prenatal and maybe early postnatal ontogenesis in mammals due to the decrease in HCN4 expression and enhancement of protein expression of working electrophysiological phenotype. In other words, the ontogenesis is accompanied by the compactization of the heart pacemaker and atrialization turns out to be both morphological and functional [48]. It has been established that the myocardium of cranial (anterior) venae cavae is characterized by depolarized and unstable resting membrane potential, low conduction velocity, and weak electric coupling between cardiomyocytes in rats at the early stages of postnatal ontogenesis, thus partially demonstrating the features of pacemaker myocardium. These features are associated with reduced expression of inward rectifier potassium current Kir2.x channels and connexins Cx43. The myocardium of venae cavae originates from the same group of cells as the cardiomyocytes of embryonic cardinal veins and SV. The myocardial sleeve of anterior cardinal veins which are actually extending of SV wall is modified during postnatal ontogenesis into the myocardial sleeve of the cranial venae cavae. The expression of Kir2.x and Cx43 enhances in venae cavae during ontogenesis. Thus, the electrophysiological properties of this tissue become closer to phenotype of working myocardium [48]. Probably, postnatal atrialization of myocardium in the venae cavae can also take place in large mammals, including humans. The localization, compaction, and isolation of myocardium capable of automaticity promote the mature SAN function during ontogenesis in mammals, i.e., the ability to rhythm generation with a frequency up to 10 Hz. On the contrary, incomplete atrialization or delayed transformation of electrophysiological phenotype can facilitate ectopic activity in the myocardium of the venae cavae and cause arrhythmias associated with congenital abnormalities.
It was shown that SV atrialization and SAN compactization are associated with the alteration in TF expression pattern in mammals, primarily, by the emergence of Nkx2.5/GATA4 expression in complex with TBX5 transcription activator in Nkx2.5-negative cells [29,50]. It was clearly demonstrated that the level of Nkx2.5 expression significantly increases, though not up to the level of working myocardium, in the myocardial tissue of the venae cavae in ontogenesis. The increased expression of the TFs is simultaneous with the formation of sympathetic innervation in rats and some other mammals, probably due to norepinephrine secretion with adrenergic fibers and their trophic effects. As mentioned above, the Nkx2.5/TBX5/GATA4 complex determines the differentiation of cardiomyocyte progenitors into working atrial myocardium [20]. It is known that the myocardium of pulmonary veins is the most common substrate responsible for ectopic activity and atrial fibrillation induction. It has earlier been demonstrated that the level of Nkx2.5 expression in pulmonary veins decreases during postnatal development in contrast with venae cavae, probably providing the higher proarrhythmic potential of the tissue due to the loss of working phenotype and enhancement of pacemaker characteristics [50]. Therefore, TFs participating in the formation of SAN also contribute to the induction of pathological processes, particularly arrhythmias.
Developing SAN appears to be protected from the program of atrialization. One of the mechanisms of such protection is the high expression of the TBX3 transcriptional repressor, which facilitates the activation of the propacemaker genetic program [51,52]. TBX3 can be considered as antagonist of Nkx2.5/TBX5/GATA4 [53,54]. TBX3-positive cardiomyocytes of the heart venous pole demonstrate automaticity unlike TBX3-negative ones. It has been found that TBX3 suppresses the expression of proteins determining the working electrophysiological phenotype in a dose-dependent manner and reprograms the working cardiomyocytes into pacemaker ones [55]. The reduction of TBX3 expression below a certain threshold leads to the differentiation of SAN progenitor cells into working cardiomyocytes. The suppression of TBX3 during cardiogenesis provokes SAN hypoplasia, bradycardia, or bradyarrhythmia in experimental animals.
Epigenetic transcription control involving TBX3 is mediated through the formation of a heterocomplex that includes TBX3, Baf250a (ARID1A) protein and one of histone deacetylases (Hdac3), which deacetylates the histones of Nkx2.5 locus, causing suppression of the latter [56]. Isl1 is considered as one of the activators of TBX3 expression (Fig. 2).
Presumably, TBX3 alone is insufficient for the formation of SAN; instead, TBX18, as already mentioned, determines the fate of SAN progenitor cells [40]. TBX18 is necessary for specification and localization of an individual clone of mesenchymal cells in mesoderm as well as for differentiation of progenitor cells into cardiomyocytes of SV and SAN. In addition, the TBX2 repressor might also contribute to the specification of SAN progenitors and suppression of working phenotype gene expression [57].
Incomplete atrialization of SV and incomplete compactization of SAN is probably related to the deficiency of Nkx2.5 expression and insufficient suppression of TBX2, TBX3, and TBX18. Such imbalance is capable of causing ectopic arrhythmogenic automaticity in the myocardial sleeve of venae cavae during pre-and postnatal development. It has been shown that the myocardium of venae cavae demonstrates pacemaker properties in response to α1-adrenergic receptors stimulation at least in adult rats [58]. Therefore, the pacemaker profile of TF expression might underlie the arrhythmogenic action of α1-adrenergic stimulation in some areas of supraventricular myocardium.

Asymmetrical Localization of SAN in Mammals
It has been supposed recently that visceral cardiogenic mesoderm is bilaterally asymmetrical as soon as the heart fields can be identified. It has been found that the posterior part of SHF contributes to a different extent into bilateral components of the embryonic heart [59]. The right-sided location of definitive SAN might be a result of asymmetrical migration of mesenchymal progenitor cells from the heart fields.
On the other hand, a part of SAN cardiomyocyte progenitors can appear in the proximal part of the left cardinal veins in the process of SV atrialization and then contribute to the left atrial wall. Such cells can be the source for the pathological left-sided heart pacemaker, persisting during the embryonic period or in subunits of calcium voltage-gated channels, respectively) specific for pacemaker SAN cardiomyocytes. Nppa, Gja1, Gja5, Scn5a, Kcnj2, and Kcnj12 genes of proteins (atrial natriuretic peptide, connexins Cx40, Cx43, Nav1.5 voltage-gated sodium channel, Kir2.1 and Kir2.2 inward rectifier potassium channels, respectively) specific for working atrial cardiomyocytes. postnatal life. Normally, the formation of the leftsided pacemaker phenotype is suppressed and the natural definitive heart pacemaker occurs in the right atrial wall. It has been shown that Pitx2c is the key TF that inhibits the development of pacemaker myocardium in the left SV horn and left atrium. This TF controls the formation of bilateral asymmetry and participates in the signaling Nodal/Lefty/Pitx2 pathway [20,60]. Pitx2c is a direct repressor of propacemaker TBX3 and Shox2 in the left atrium [61].
3. MOLECULAR CONTROL OVER GENETIC PROGRAMS OF SV AND SAN EMBRYOGENESIS Different structures function as pacemaker at various stages of ontogenesis, including the caudal part of the primary heart tube, SV or its regions, and finally the definitive SAN in mammals. The genetic and transcription control of heart pacemaker ontogenesis is associated with complex epistatic or network interaction between numerous factors determining the activation of the myogenic program in mesenchymal precursors, the regulation of their migration and proliferation, differentiation, and control of their electrophysiological phenotype. The same factors can regulate cardiogenesis at the earliest stages of embryogenesis, cardiogenic tissue induction, and terminal differentiation of cardiomyocytes in the prenatal period, complicating the investigation of molecular mechanisms of spatiotemporal control of the development of pacemaker structures. Nevertheless, the role of several individual signaling molecular pathways has been identified as shown further in this review.

Nkx2.5/GATA4/TBX5 and Shox2 Transcription
Factors in the Control over the Development of SV and SAN Nkx2.5 is a homeodomain protein and functions as a dimer complex with GATA4; Nkx2.5 and GATA4 are reciprocal coactivators [62]. Apart from GATA4, the functioning of Nkx2.5 is controlled by a number of T-box protein family members. TBX5 physically interacts with Nxk2.5 producing activation Nkx2.5/GATA4/TBX5 complex [63]; the complex induces the transcription of working phenotype gene in cardiomyocyte progenitors. On the contrary, TBX3 replacing TBX5 in a Nkx2.5/GATA4 complex suppresses the transcription of working phenotype genes [38]. It has been demonstrated that Nkx2.5 and Isl1 also antagonize each other and suppress each other's expression.
Nkx2.5/GATA4 controls the gene of Nppa atrial natriuretic peptide. Atrial natriuretic peptide is only expressed in those cardiomyocytes that contain a high level of Nkx2.5/N structures originating from SV express Nppa, including SAN. This peptide was con-sidered as a marker of working atrial and ventricular myocardium [64].
SV cells throughout the embryonic development as well as cardiomyocytes of definitive SAN express homeodomain Shox2. Moreover, Shox2 expression is limited to SV during the embryogenesis and SAN myocardium in adult mammals. Shox2 mutants demonstrate a substantial SAN hypoplasia accompanied by severe bradycardia. The expression of Nkx2.5 and Cx43 connexins typical for working myocardium can be observed in the intervenous area of the right atrium, corresponding with the location of SAN in Shox2 -/mutants [65]. Also, Shox2 is necessary for the formation of mesenchymal SAN progenitors.
Genes of the key TFs like Isl1, TBX3, TBX18, TBX5, and BMP4 that determine the pacemaker phenotype of cardiomyocytes are the direct Shox2 targets [66]; Shox2 acts as repressor for Nkx2.5 in SV myocardium [67]. The antagonistic balance of interacting Shox2 and Nkx2.5 has been shown to determine the phenotype of cardiomyocytes and ability of myocardial tissue to reveal automaticity. It is hypothesized that Shox2 averts SAN atrializaiton along with TBX3 and TBX18 and, at the same time, underlies the ectopic arrhythmogenic activity in the myocardium of pulmonary veins promoting the propacemaker genes [68,69]. Therefore, Shox2 is at the top of the hierarchy of TFs regulating the pacemaker (epi)genetic program [70] (Fig. 2).

Myocardial Morphogens of BMP Family
Bone morphogenic proteins (BMPs) belong to the superfamily of transforming growth factor and regulate the development of almost all tissues and organs in animals. The proteins of this family and their membrane receptors are required both for early and late stages of cardiogenesis. In particular, BMPs are involved in the differentiation of cardioblasts from mesenchymal precursors [71]. Undoubtedly, the BMPs play significant role in cardiac pacemaker development. However, only a few members of this morphogen group have a verified role in SAN formation due to numerous members in the family and sophisticated character of their interactions.
BMP2 stimulates EMT promoting the accumulation of a pool of mesenchymal cardiogenic cells and activates the expression of transcriptional repressors specific for pacemaker myocardium, i.e., TBX2 and TBX3 [45,72]. BMP2 suppresses the expression of Scn5a encoding the Nav1.5 sodium channel typical for the working myocardium [3].
BMP4 is a direct target of Shox2; this TF interacts with the Bmp4 promoter in the venous pole of tubular heart [73]. BMP4 expression is almost entirely suppressed in Shox2 -/mutants; furthermore, HCN4 expression is reduced and Cx40 expression is excessively distributed within the dorsal mesenchyme in the mutants [47]. BMP4 stimulates the differentiation of TBX18-positive precursors into myocyte-like cells that exhibit pacemaker action potentials at least in embryonic culture [74]. These data suggest that BMP4 is crucial morphogenic factor for SAN.

Signaling Proteins of the Wnt Family; Wnt/β-Catenin and Pathways in SAN Morphogenesis
The class of secreted Wnt proteins includes over ten members in mammals; Wnt proteins participate in the maintenance of cells stemness, intercellular communication, mesoderm induction and regulate embryogenesis in a number of organs and tissues [75]. Wnt signaling pathway includes G protein-coupled receptors of the Frizzled family (Fz receptors) [76]. Wnt signaling is strictly regulated by numerous factors, such as inhibitors of Wnt signal transduction, of which DKK1 (dickkopf-related protein family) is mostly known of them. Wnt signaling pathways are divided into two types: canonical and noncanonical. The activation of Fz receptor leads to the stabilization of cytoplasmic β-catenin in the canonical signaling pathway. Β-catenin accumulating in the nucleus binds and activates the TCF/LEF complex, a TF for Wnt target genes [77]. One of the noncanonical Wnt pathways involved in cardiogenesis includes JNK kinase (c-Jun kinase). JNK kinase activates c-Jun protein that forms a heterodimeric immediate response transcription factor AP-1 (activator protein 1) together with the c-Fos protein [7].
The variety of ligands in Wnt signaling pathways, along with the complexity of spatiotemporal pattern of Wnt element expression, complicate the investigation of the role of this regulatory factor in the morphogenesis of heart structures. Nevertheless, it has been found that signaling mediated both via the canonical Wnt/β-catenin (Wnt/β) and noncanonical Wnt/JNKpathways at the early stages is necessary for the induction of Isl1-and Nkx2.5-positive cardiogenic mesoderm, maintenance of pool and proliferation of cardiogenic precursor cells, and for prevention of premature differentiation of mesenchymal cells to cardiomyocytes [78].
After the segregation of heart fields, Wnt/β-catenin signaling restricts the differentiation and stimulates the proliferation of Nkx2.5-negative cells of SHF. Therefore, Wnt/β facilitates the cell recruitment and development of the venous heart pole (SV and its horns), probably via Wnt2 and/or Wnt10a, and provides myocardilization of cardinal veins [79]. On the contrary, noncanonical Wnt/JNK signaling stimulates terminal differentiation of cells originating from all heart fields (Fig. 3a) [78,80]. At least one study showed that manipulations suppressing Wnt/β or Wnt/JNK do not affect TBX18-positive cell pool formation in SV and further formation of SAN [79]. These data confirm the assumption that SAN precursor cells separate from the cardiogenic mesoderm very early, while Wnt acts independently from TBX18 and participates only in embryogenesis of the proper environment for migrating SAN precursor cells (cardinal veins).

Wt1 Protein
The product of the Wt1 gene is a TF previously known as a protein involved in the pathogenesis of Wilms nephroblastoma (Wt1-Wilms tumor 1). It has been established that Wt1 is crucial for the morphogenesis of several organs. This TF stimulates the recruitment of mesenchymal progenitors from cardiogenic mesoderm by EMT in cardiogenesis, thus suppressing E-cadherin expression. The suppression of E-cadherin Cdh1 gene expression is associated with the enhancement of Snail transcription encoding the gene repressor of adhesion proteins [81]. Wt1 is pivotal for normal development of common cardinal veins, their myocardilization, and formation of SV horns [82]. Wt1 is needed for correct localization of cardinal veins in the caudal region of the heart tube (Fig. 3a). Interestingly, SAN precursor cells can be found in Wt1 -/mutants, however, the formation of definitive SAN is impossible.
Presumably, the effects of Wt1 are associated with local stimulation of Raldh2 expression, a gene of retinaldehyde dehydrogenase 2 synthesizing the retinoic acid (RA) from retinaldehyde. RA is a factor stimulating cardiomyocyte differentiation [83] and local apoptosis of the mesenchymal cell group, thus promoting the correct positioning of common cardinal veins and the formation of substrate for future SAN.

Slit/Robo Signaling Pathway
The proteins of the Slit family are secreted extracellular molecules with the control of axonal growth as the most known function [84]. Proteins of the Robo family, or the receptors of roundabout family, are membrane Slit receptors. The expression of Robo1-4 identified in mammals [85,86]. Robo receptors interact with cytoplasmic proteins involved in the pathways regulating cell adhesion, proliferation, and cytoskeleton modifications [87].
It has currently been shown that the ligands of the Slit family (Slit2 and Slit3) and their Robo receptors (Robo1 and Robo2) are expressed in the promyocardial SHF cells within the cardiogenic crescent, cardiomyocytes of the primary heart tube, and different heart compartments at the later stages of prenatal ontogenesis [87], thus making a substantial contribution to cardiogenesis. The mutations in the genes of the Slit/Robo signaling pathway provoke various abnormalities of heart development. In particular, Robo gene mutations are associated with tetralogy of Fallot and Holt-Oram syndrome [87,88].
Since Slit2 expression was found in mesenchyme surrounding venae cavae and Slit3 expression was detected in the myocardium of SV horns, it was proposed that the Slit/Robo signaling pathway participated in the formation of SV and SAN [89]. Moreover, Robo1 and Robo2 receptors are expressed around the venous pole of the primitive heart and mesenchyme of the posterior SHF region [90]. SV horns fail to develop in Robo1 -/and Robo2 -/double mutants, leading to hypoplasia of venae cavae (Fig. 3b) [87]. However, the accumulation of SAN cells in mentioned mutants remains intact [89]. This fact supports the theory of early separation of SAN progenitors and SV myocytes during embryogenesis. Furthermore, no myocardilization occurs in the cranial superior venae cave in Slit3 -/mutants. Potentially, the altered activation of the Slit/Robo cascade can affect the arrhythmogenicity of the myocardial sleeve of venae cavae.
The regulation of Slit/Robo signaling pathway is closely related to the key TFs of cardiogenesis, i.e., TBX2, TBX20, TBX5, and Nkx2.5. TBX2 TF is able to bind the Slit3 gene promoter, thus restricting its expression; Nkx2.5 also inhibits the expression of Slit genes; conversely, TBX20 stimulates Slit expression. Presumably, TBX2, 20, and Nkx2.5 TFs determine the spatial pattern of Slit expression in the growing heart in a complicate manner [87,90].
According to the aforementioned, cardiogenic mesenchyme of the posterior heart field and the lateral rim is formed via recruitment of visceral mesoderm cells, which undergo EMT. These cells can be considered as a coelomic epithelium, or mesothelium cells. To initiate the EMT program, the cell adhesion proteins like E-cadherins expressed in mesoderm should be inhibited. Podoplanin suppresses E-cadherin expression, facilitating EMT [92], migration, and expansion of mesenchymal cardiomyocyte precursors of the heart's venous pole. The inhibition of podoplanin expression in the embryogenesis causes SAN hypoplasia [93,94].
Podoplanin activates the RhoA/Rock signaling pathway, including small GTPase of the Rho family (RhoA) and Rho-associated protein kinase (Rock), respectively. It is hypothesized that proteins of the ERM group (ezrin, radixin, and moesin) interact with podoplanin, supporting the connection between plasmamembrane and cytoskeleton and determining the cell motility [95]. The interaction between podoplanin and ERM proteins results in the activation of RhoA, facilitating EMT in SHF mesothelium. It has been shown that RhoA activation inhibits the expression of E-cadherin, thus stimulating EMT and accumulation of precursor cells, as well as SAN development (Fig. 3c) [93].

Small RhoA/Rock GTPase Signaling Pathway
The small RhoA/Rock GTPase signaling pathway is known as a central regulator of dynamic cytoskeleton reorganization. Besides, RhoA/Rock is involved in the control of migration, proliferation, and differentiation of cells in the ontogenesis [96].
It has recently been show that RhoA/Rock plays an essential role in early cardiogenesis and formation of the cardiac pacemaker. Initially, RhoA is expressed throughout the heart tube, though it is gradually restricted to a small Nkx2.5 -/Isl1 -/TBX18 + -cell group in the course of ontogenesis, i.e. corresponds to the localization of SAN progenitors at least in chicken embryos [97]. The suppression of RhoA/Rock signaling and inhibition of Rock expression causes the reduction of TFs crucial for SAN in mammals, particularly, Isl1, TBX3, and Shox2. At the same time, the suppression of RhoA/Rock induces the aberrant pattern of TF expression, i.e., high Isl1 and Shox2 levels outside the right SV horn. Moreover, experimental disruption of the RhoA/Rock signaling cascade led to the appearance of pacemaker action potentials outside the zone of primordial SAN as well as in the left SV region; abnormal activation and conduction pattern was observed in the SV (Fig. 3c) [98].
In conclusion, RhoA/Rock is crucially important for the formation of definitive SAN; the activity of the signaling pathway is important for the lateralization and compaction of SAN and is probably mediated by controlling the TFs crucial for SAN. Besides, RhoA/Rock restricts the expression of propacemaker genes outside definitive SAN, thus preventing ectopic arrhythmogenic activity in the atrial myocardium.

Vascular Endothelial Growth Factor
A number of studies have shown that overexpression of one of the proteins (VEGF 120 ) from the vascular endothelial growth factor family (VEGF) causes bradycardia and SAN hypoplasia with reduced density of cardiomyocytes in the pacemaker area and enhanced vascularization [99,100]. In addition, excessive expression of VEGF 120 results in upregulation of Cx43 connexins in SAN and its malfunctioning [101]. Therefore, there is an experimental proof of the VEGF role in SAN formation.
One of the signaling pathways activated by VEGF via VEGFR2 receptors is a so-called Dll4/Notch-cascade [102]. Transmembrane Notch proteins, the products of genes initially associated with the mutation manifesting in concave serration at the most distal end of the wings of drosophilas, together with Dll4 (deltalike protein 4), are the most important mediators of intercellular interactions. Signaling mediated by Dll4/Notch regulates the migration and proliferation of cells in the development. The activation of VEGFR causes the translocation of intracellular domain of the Notch protein inside the nucleus, where it acts as transcription coactivator. Despite the fact that the VEGF/Dll4/Notch signaling pathway is considered as mostly endothelial, or endocardial, it has been found that this cascade coordinates the differentiation and proliferation activity in SHF [101]. At the late stages of cardiogenesis, VEGF/Dll4/Notch regulates trabecularization of myocardium and development of cardiac valves and coronary arteries [103].
It has been established that Notch1 is pivotal for normal SAV development, specification of precursor cells, and SAN formation. The suppression of Notch1 expression in endocardium terminates the development of SAV and reduces the amount of cells expressing HCN4 and TBX18 and leads to SAN hypoplasia. Notch was shown to be necessary for the induction and recruitment of mesenchymal TBX18-positive SAN progenitors as well as for proliferation of this cell clone (Fig. 3d). It is thought that neuregulin is the mediator of endocardial Notch1 factor in the promyocardial mesenchyme, due to the activation of the canonical Wnt/β-catenin pathway [104].

Platelet-Derived Growth Factor
Similar to VEGF, the most known role of plateletderived growth factor (PDGF) is the stimulation of angiogenesis. The effects of PDGF are mediated by the activation of membrane receptors with tyrosine kinase activity. It has been found that PDGF and its Pdgfr-α receptor are required for the specification of procardiogenic cells of posterior SHF, i.e., the mesodermal area possibly containing SAN precursors [105]. There are controversial data on the role of PDGF/Pdgfr-α in late cardiogenesis and SAN formation. According to the results of early studies, the suppression of Pdgfr-α expression leads to SV and SAN hypoplasia. The levels of Nkx2.5 and Wt1 have been reported to be increased in different areas of the developing heart, including cardinal veins and SAN in Pdgfrα -/mutant mice [106]. However, according to the later studies, no morphological or functional changes of SAN were observed in Pdgfrα -/mutants [107]. The Pdgfr-α receptor is probably necessary for the accumulation of the mesenchymal SAN precursor cell pool in cardiogenic mesoderm and control of their migration at the earliest stages of ontogenesis (Fig. 3d).
The latest analysis of transcriptome of fetal cells and cardiomyocytes in mature SAN using RNA sequencing showed that the expression of over 2000 genes, including the genes of TFs, ion channels, and calcium handling proteins was substantially elevated in pacemaker cells compared to cardiomyocytes of working myocardium.
Significantly increased expression can be seen in SAN cells for Notch and its functionally associated genes; genes related to the BMP signaling cascades; genes associated with the organization and remodeling of extracellular matrix and intercellular junctions. These data indirectly prove the involvement of a number of aforementioned regulatory cascades in the formation of the heart pacemaker in mammals. It has also been demonstrated that the expression of the key TBX3, Isl1, Shox2, Hcn4, and BMP signaling genes remains elevated not only in the embryonic period of ontogenesis but also in cardiomyocytes of definitive SAN, supporting the unique electrophysiological phenotype of pacemaker cells.
Therefore, the main molecular regulators necessary for the formation of SAN have currently been identified. More important factors include those inducing the cardiogenic mesoderm and EMT, promoting the accumulation of mesenchymal SAN precursor cell pool (BMP, Wnt, and VEGF) and stimulating their migration (podoplanin). Secondly, the essential factors should include those facilitating the induction of pacemaker electrophysiological phenotype in SAN precursor cells, i.e., TBx3, TBX18, and Isl1, while Shox2 is potentially the most significant of them.
The determination of molecular factors directing the fate of the precursor cells towards the formation of pacemaker cardiomyocytes resulted in certain positive advances in elaborating the bio-artificial pacemakers. As mentioned above, the enhancement of propacemaker TF expression in the working mature cardiomyocytes or induced pluripotent stem cells (iPSCs) is capable of reprograming them into pacemaker-like cells [55,108,109]. It should be noted that the main approach for automaticity induction in cardiomyocytes has been to transduce cells or myocardial tissue with vector constructions that suppress the expression of inward rectifier potassium channels (Kir2.x) or enhance the expression of HCN channels, responsible for I f pacemaker current until recently. As shown in many studies, that transduced cardiomyocytes indeed gain the capacity for spontaneous generation of action potentials, though no appropriate SAN cell phenotype is acquired.
The progress in cardiac cells reprogramming is considerably confined to the specification of content in regulatory factor cocktails, either inhibitors or upregulators of the receptors, used to guide the differentiation of iPSCs, embryonic stem cells, or embryonic cardiomyocytes into the pacemaker cells. Modern protocols utilizing the inhibitors of Wnt, BMP, and RhoA/Rock signaling pathways provide a substantial outcome of SAN-like cells in the population iPSCderived cardiomyocytes [110]. Such SAN-like cells demonstrate automaticity, pacemaker action potentials, can be Nkx2.5-negative, and express Isl1, TBX3, TBX18, Shox2, HNC4, and Cx45. Nevertheless, the populations of SAN-like cells obtained from iPSCs are highly heterogenic both in electrophysiological properties and protein expression profile, complicating their application in cell-based therapy.
Bioengineering of biological pacemaker-like organoids, cell-based therapy or local control of gene expression for heart rhythm recovery are widely discussed in literature [111]. Presumably, stepwise manipulation of TF expression is the perspective method for obtaining of SAN-like cells from iPSCs, for the directing the development and differentiation into pacemaker cells. It has been shown that time-controlled stimulation with BMP4, FGF, and activin А, followed by the treatment with the inhibitors of Wnt and VEGF production leads to almost full differentiation of iPSCs into SAN-like cells expressing Isl1, TBX3, TBX18, Shox2, HNC4, and Cx45 [112]. It has been experimentally proven that such SAN-like cells show pacemaker activity after transplantation into working ventricular myocardium, thus forming a bioartificial pacemaker.
Along with the abovementioned, transdifferentiation of somatic cells is considered perspective, specifically of fibroblasts or mature working cardiomyocytes, into SAN-like cells [111,113]. A number of studies have shown that the induction of expression of one or several propacemaker TFs, e.g., TBX3 [55], TBX18 [114], and Isl1 [115], leads to reprogramming of somatic cells into cardiomyocytes demonstrating SAN phenotype and ability to maintain rhythm after transplantation into working myocardium to a greater or lesser degree.
In conclusion, it should be noted that transcriptome profiling revealed a significant amount of new SAN-specific factors that were not earlier known to participate in the development of the heart pacemaker [3,116]. As a result, versatile and sophisticated regulation of heart pacemaker development in vertebrate animals still requires further investigations, despite the substantial progress in understanding of the morphogenesis, the localization of major cell population, and identification of pivotal TFs.

COMPLIANCE WITH ETHICAL STANDARDS
Conflict of Interests. The authors declare that they have no conflict of interests.
Statement on the Welfare of Animals. This article does not contain any studies involving animals or humans performed by any of the authors.