HomeCirculation ResearchVol. 82, No. 6Development of the Cardiac Conduction System Free AccessOtherPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessOtherPDF/EPUBDevelopment of the Cardiac Conduction System Antoon F.M. Moorman, Frits de Jong, Marylène M.F.J. Denyn, and Wouter H. Lamers Antoon F.M. MoormanAntoon F.M. Moorman From the Cardiovascular Research Institute Amsterdam, Academic Medical Center, University of Amsterdam (the Netherlands). Search for more papers by this author , Frits de JongFrits de Jong From the Cardiovascular Research Institute Amsterdam, Academic Medical Center, University of Amsterdam (the Netherlands). Search for more papers by this author , Marylène M.F.J. DenynMarylène M.F.J. Denyn From the Cardiovascular Research Institute Amsterdam, Academic Medical Center, University of Amsterdam (the Netherlands). Search for more papers by this author , and Wouter H. LamersWouter H. Lamers From the Cardiovascular Research Institute Amsterdam, Academic Medical Center, University of Amsterdam (the Netherlands). Search for more papers by this author Originally published6 Apr 1998https://doi.org/10.1161/01.RES.82.6.629Circulation Research. 1998;82:629–644Basic ConceptsIn the formed heart, it is convention to distinguish working myocardium (the primary function of which is contraction) from the conduction system (the primary function of which is the generation and conduction of the electrical impulse). The conduction system comprises separate components with distinct functions. The SAN, which contains the leading pacemaker, generates the impulse. The impulse is subsequently conducted, via the atrial myocardium, which in this sense is part of the conduction pathway as well, toward the AVN. With a delay, the impulse is then rapidly transmitted from the AVN via the bundle branches and PPN to ensure a coordinated activation of the ventricular myocardium from apex to base. Classic reports cover the anatomy,1 pathology,1 and histology2 of the adult and developing conduction system.The myocytes of the conduction system share with those of the ordinary working myocardium four basic elements: (1) contraction, (2) autorhythmicity, (3) intercellular conduction, and (4) electromechanical coupling. In the early embryonic heart tube, an ECG, similar to an adult ECG, can be recorded, indicating the presence of sequentially activated chambers.3 Given this observation, it is as confusing to accept the presence of a conduction system because it is functionally present as it is to deny its existence because it is not morphologically recognizable. Rather, it is of paramount importance to appreciate that the arrangement of myocyte populations, with distinct contractile, conductive, and pacemaking properties, establishes the coordinated activation of the heart. Departures from these tenets have led to a confusing and fruitless search for so-called “cardiac specialized tissues” during development. The obvious key question is how this arrangement is being achieved.Early cardiac development starts with the formation of a primary heart tube from the cardiogenic mesoderm (Fig 1); this topic has been reviewed recently.4 The primary heart tube is a peristaltic pump that moves blood ahead as a result of a unidirectional wave of contractions along the tube. Within this slow-conducting heart tube, fast-conducting and synchronously contracting atrial and ventricular chambers develop; these chambers remain flanked by the slow-conducting primary myocardium of the IFT, AVC, and OFT (Fig 2).5 The configuration of alternating slow- and fast-conducting segments guarantees that the downstream ventricular segment does not contract before the termination of the contraction of the upstream atrial segment and is responsible for the embryonic ECG. This configuration ensures also that relaxation of the atrial or ventricular segment does not occur before contraction of a downstream flanking segment, by which regurgitation of the blood is prevented. The sphincter-like prolonged peristaltic contraction form of the slow-conducting flanking segments substitutes for the adult type of one-way valves; this phenomenon is essential in a heart in which atrioventricular and semilunar valves have not yet developed.6 The slow-conducting SAN and AVN will take origin from the slow-conducting myocardium of IFT and AVC, sometimes referred to as “cardiac specialized tissues.” The fast-conducting AVB, bundle branches, and their ramifications will develop from the ventricular segment. This building plan of early cardiac development in modules provides the framework to structure the anecdotal facts on the developing conduction system. Our aim here is to make evident that a consequent morphological, functional, and molecular description of the development of the cardiac tube and its constituting segments encompasses, of necessity, the “conduction system.”Development of the NodesDevelopment of PolarityOne of the most striking features of the cardiogenic mesoderm and of the subsequently formed cardiac tube is its polarity along the anteroposterior axis. This is not yet the case in our chordate ancestors, the tunicates, in which the position of the leading pacemaker activity is not fixed and thus the heart pumps blood in both directions.7 The polarity of the vertebrate heart is characterized by the predominance of the atrial phenotype posteriorly (at the inflow/upstream side of the heart) and of the ventricular phenotype anteriorly (at the outflow/downstream side of the heart).5 Although at both extremities of the heart tube, myocardium is added,891011 dominant pacemaker activity312 and highest beat frequency121314 are invariably found at the intake of the tube, by which an efficient contraction wave is always ensured. The observation that the first contractions are observed in the middle (ventricular) part of the cardiactube corresponds with the finding that excitation-contraction coupling is first achieved in the ventricular portion. Thus, there is no pacemaker jump from the ventricular portion toward the venous pole of the heart during development.3 Cells of the future sinus region show prepotentials resembling those of the adult pacemaker, and cells of the cardiac tube, the future ventricles, show an electrical behavior similar to that of adult ventricles.1516The early development of conduction in the heart has been studied mainly in avian embryos.1617 When 7 to 10 somites have developed (equivalent age of a human embryo of ≈20 days), a single pacemaking area becomes established at the IFT of the heart.18 Pacemaker dominance increases along the anteroposterior axis.312 Also, the frequency of the intrinsic beat rate increases along this axis.121314 In both birds and mammals, the leading pacemaker area is initially found on the left side,14192021 but as soon as the sinus venosus has formed (≈25 days in humans), the right side starts to become dominant. The flexibility of the nodal locations may not be surprising, if one takes into account the dimensions of the inflow area at this stage of development and the much bigger adult SAN, in which the leading pacemaker site is not fixed either.2223 In addition, both right and left IFTs will become incorporated into the right atrium. Thus, in fact, the entire inflow area represents a more or less homogeneous pacemaking area, where the left side predominates. In line with this notion is the observation of node-like cells in the myocardium surrounding the distal portion of the pulmonary veins in adult rat.24 These cells appear to guarantee a unidirectional flow of blood into the left atrium and to prevent regurgitation of the atrial blood into the pulmonary veins.2526The molecular signals that impose polarity on the cardiac tube are unknown. Transplantation experiments of cardiomyocytes to another position along the anteroposterior axis of the early embryonic chicken heart have demonstrated that cardiomyocytes initially have the capacity to adapt their phenotype according to the new position.1227 Retinoic acid induces a posteriorization of the phenotype in the anterior portion of the heart.2829 These data demonstrate that initially cardiac polarity is not fixed.In humans and other mammals, the first morphological signs of the SAN are at Carnegie stage 15 (≈5 weeks of human development) in the anteromedial wall of the right common cardinal vein, giving rise to the superior caval vein.3031 In chickens and lower vertebrates, it remains a loosely arranged conglomerate of venous sinus myocytes.2323334 How the leading pacemaker area in mammals becomes transformed into a node that is morphologically and molecularly distinct from the surrounding atrial working myocardium and what the nature is of the molecular signals have remained enigmatic so far.In conclusion, from the beginning onward, polarity is present along the cardiac tube, with the leading pacemaker (SAN) being present at the most posterior part of the developing tube, which guarantees the unidirectional wave of contraction.Chamber Formation and the Development of an ECGA fundamental property of the primary heart tube is the slow conduction of the impulse,63536 resulting in a slow and peristaltic contraction,37 paraphrased by Patten and Kramer38 as “a progress of the contraction wave along the cardiac tube as striking and characteristic as intestinal peristalsis.” We have dubbed this early slow-conducting myocardium the “primary myocardium,” as opposed to (atrial and ventricular) working myocardium.5 Among other things, the primary myocardium is characterized by action potentials, which display slow depolarizations reminiscent of pacemaker action potentials and typical of slow voltage-gated calcium ion channels.3940With further embryonic development, atrial and ventricular chambers that contract synchronously and sequentially begin to develop (Fig 2).3738 This is accompanied by the development of an adult type of ECG, which merely reflects the sequential activation of the atrial and ventricular chambers rather than the presence of a morphologically recognizable conduction system.341 The synchronous contractions of the atrial and ventricular working myocardium indicate that these segments are characterized by the development of high-conduction velocities.6354243 Concordantly emerging are action potentials, which have a fast rising phase and high amplitude characteristic of fast voltage-gated sodium ion channels.3940 Paff et al44 concluded that in the embryonic chicken heart after 42 hours of incubation (≈25 days of human development) “it would appear that a conduction system consisting of a pace-making sinuatrium, atrioventricular junctional tissue, ventricle and conus regions is developing.” Thus, the authors consider the entire embryonic heart as a conducting unit without the presence of a morphologically identifiable “adult conduction system,” which is in line with the view of Patten,45 who concluded from experimental studies that “the whole of the primary myocardium constituting the wall of the myocardial tube was acting as a conducting tissue.” It should be noted as well that an atrioventricular delay has developed before the development of a morphologically identifiable AVN.4647 The AVC was recognized as the zone of slow conduction.363541424348 Therefore, it functions as the “AVN equivalent” in a heart in which atrial and ventricular myocardial masses have not yet been insulated by fibrous tissue. In fact, the AVC represents “primary myocardium” remaining between the atrial and ventricular chambers.6Segments of slow conduction (remaining primary myocardium) also persist at both extremities of the heart.649 Paff and coworkers5051 have already concluded that the OFT remains the least differentiated part (compare with “primary myocardium”5 ) of the tubular heart50 and that “the prolonged contraction of the OFT produces a sphincter-like closure of the OFT at the end of systole. Thus without valves, little regurgitation of blood occurs.”51 Later, Paff and Boucek49 reported delayed contractions of the OFT and delayed propagation of the impulse from ventricle to OFT as C waves in the ECG. Finally, de Jong et al6 recorded slow conduction in all three flanking segments (IFT, AVC, and OFT), the functional significance of which has been pointed out above (“Basic Concepts”). The sphincter function of the OFT is in part retained in the formed dog heart, where it has been shown that the musculature surrounding the right ventricular OFT maintains the normal tonus during ventricular relaxation and so provides the necessary support for the pulmonary semilunar valves.52The AVN as a nodal structure becomes only gradually identifiable from about Carnegie stage 15 (≈5 weeks of human development) onward.5354 In the chicken, it remains an indistinct entity, and the atrioventricular junction has been supposed to fulfill a role similar to that of the AVN.34 It is of great interest that in dog and pig hearts the entire lower rim of the left and right atria just above the fibrous annulus, ie, the former AVC, still has “nodal characteristics,” based on the presence of nodal-like action potentials and low abundance of the gap-junctional protein Cx43 and its encoding mRNA.555657In summary, with the process of chamber formation, fast-conducting atrial and ventricular segments are being formed within the slow-conducting primary myocardium of the embryonic heart tube, so that the cardiac tube becomes a composite of alternating slow- and fast-conducting segments. ECGs show that this arrangement of segments provides the embryonic heart with an “electrical architecture” similar to that in the formed heart. The molecular basis underlying the compartmentalization is beginning to emerge through the analysis of the developmentally regulated patterns of transcription factors4 and of the modular patterns of expression of the lacZ transgene under direction of various cardiac promoter constructs.58Development of the Nodal PhenotypeIn the formed heart, the nodal myocytes are said to display a number of “embryonic characteristics.”25960 Similar to embryonic cardiomyocytes, nodal myocytes are small compared with the myocytes of the surrounding atrial working myocardium, and they have poorly organized actin and myosin filaments and a poorly developed sarcoplasmic reticulum.2 Therefore, in early embryonic hearts they can hardly be distinguished from the surrounding myocardium by unique histological characteristics.2305361 Instead, they initially are indicated by their separate arrangement and topography, which have obviously been the cause of much controversy. The signals that lead to the so-called “aggregation of the nodal area” are unknown. Innervation that occurs in the same time period30 may play a role, but its significance to nodal development has yet to be assessed. When the atrial working myocardium differentiates, the nodes become more easily identifiable because the nodal myocytes remain “primary” in many aspects (see below).So far, a universal description of the “nodal molecular phenotype” is lacking, indicating that many phenotypical features are not restricted to the nodal myocyte. However, several classes of genes display a pattern of expression that allows, within a species, the distinction of the nodal myocytes from the surrounding atrial working myocardium. Data are summarized in Fig 3. Added to the complexity is the interspecies variability, not only with respect to the type of genes expressed and the level of expression (eg, the interspecies variability of desmin expression626364 ) but also with respect to the number and pattern of the nodal cells that express the gene. This has hampered the interpretation of the functional significance of the patterns of gene expression considerably and the general use of these genes as markers to delineate the nodes morphologically.ConnexinsA crucial characteristic of cardiogenesis is the development of alternating slow- and fast-conducting segments. Gap junctions are held to be responsible for the intercellular transfer of the depolarizing action potentials in the myocardium.65 Gap junctions are aggregates of membrane channels composed of protein subunits, dubbed connexins, that are encoded by a multigene family.66 Five different connexins are expressed in the mammalian heart (Cx37, Cx40, Cx43, Cx45, and Cx46). In the early myocardium, both the number and size of gap junctions is small but increases during development.566768 However, gap junctions remain scarce in the developing SAN and AVN.304856 Cx40 and Cx43 protein and mRNA57 were found to be undetectable in the flanking segments (OFT, AVC, and IFT) and the developing nodes and to be rare or undetectable in the adult structures of the rat,56576970 guinea pig,7071 pig,71 cow,717273 and human717475 (Fig 4). In human SAN and AVN tissue, the small and scarce gap junctions display faint expression of Cx40 and Cx45.75 The low abundance of connexin expression in the nodes corresponds with the slow conduction velocities observed in the nodes and the absence of fast sodium currents.76 The poor coupling of the nodal cells appears to be a requirement to prevent silencing of the pacing nodal myocytes by the much bigger atrial/ventricular working myocardium.777879The low abundance of connexin expression in the nodes has been very useful, in conjunction with the use of node-specific intermediate filament markers, in delineating the interdigitation of nodal and atrial myocardium.7273 Cx43 and the intermediate filaments display an almost mutually exclusive pattern of expression in the atrial and nodal myocardium, respectively; ie, an abrupt rather than a gradual increase in the number of gap junctions is found at the transitions of the nodal tissue to the atrium. Consequently, a gradient in the molecular phenotype of the nodal myocytes may not be the explanation for the proposed gradient in resistivity that is essential for the pacemaker function of the SAN.77 Instead, the electrical gradient seems to be the result of a gradual change in the morphology of the nodal cell toward its periphery and a decrease of the number of nodal cells toward the atrial working myocardium rather than a gradient in molecular phenotype.80Contractile ProteinsWhereas the functional significance of specific connexin isoform expression in the nodes can be envisioned, this is more problematic in the case of the so-called persistent expression of genes encoding “embryonic and/or skeletal” contractile protein isoforms, which are often mentioned as characteristic of the conductive tissues.The expression of the myosin isoforms starts before contraction is being observed, as shown in the chicken, where coexpression of α- and β-MHC has been described.81 With the confinement of the expression of α- and β-MHC to the atrial and ventricular working myocardium, respectively, coexpression of the MHCs becomes characteristic for the nodal areas. In the SAN, coexpression of the MHCs is a common feature in a wide variety of species, including chickens,33 rats,82 cows,83 and humans.3184 In chickens, both MHCs are coexpressed throughout the entire node, whereas in mammals, the β-isoform is expressed at the rim of the SAN only. Also, the atrioventricular nodal cells coexpress both myosin isoforms in chickens,4785 cows,86 and humans.8487 In the developing AVN of rats82 and mice (authors’ unpublished data, 1997), however, no expression of β-MHC was found, which may be a characteristic of small animals or merely reflect phylogenetic interspecies variation.In cows, a nodal myosin isoform immunologically related to the fetal skeletal myosin isoform has been observed in the nodal tissues but not in the Purkinje fibers of the ventricular conduction system.8388 The atrioventricular nodal cells that were positive for the skeletal fetal MHC antibody did not reveal immunoreactivity for β-MHC.83 Also, in rats, a MHC isoform related to embryonic skeletal MHC has been localized in the nodal regions from 13.5 days of development onward.89 In contrast to the bovine heart, where the expression of the fetal skeletal MHC isoform persists,83 the expression of the rat fetal isoform decreases a few days after birth. Unfortunately, the antigen could not be visualized on Western blots of cardiac protein extracts.In rats, expression of the major embryonic form of troponin I persists in the adult AVN.9091 This embryonic troponin I isoform is identical to the isoform expressed in slow skeletal muscles.92 The mRNA can be visualized in hearts from 10 days of development onward and decreases after birth. These findings are underscored by the observation that 4200 nucleotides of the upstream sequences of the human slow skeletal troponin I gene are able to confer expression of the reporter gene to the adult mouse AVN.93 The onset of expression of cardiac troponin I occurs later in rat heart development (11 days of development) and persists in the entire adult heart. Hence, transiently, the fetal myocardium displays coexpression of both isoforms, similar to the adult AVN.The functional significance of the expression of the slow β-MHC isoform, of the fetal skeletal myosin isoform, and of the slow skeletal troponin I isoform in nodal cells remains unaccounted for. It may be the obligatory consequence of the nodal (“embryonic”) program of gene expression.Cytoskeletal ProteinsDesminDesmin, the major component of the intermediate filaments in Purkinje fibers,62 is already expressed in early mouse and rat myocardium.6494 It has been shown that specifically phosphorylated desmin isoforms are present in the conduction system (nodes, bundle, and bundle branches) of the adult cow.95 As mentioned above, several monoclonal antibodies were raised against desmin-like proteins, reacting specifically with the bovine conduction system and allowing the delineation of the conduction system.727396 The high abundance of desmin in the conduction system has led to the suggestion that it could play a role in the reduction of the changing mechanical stress during systole and diastole in the myocytes of the conduction system.6263 In a study in which desmin immunoreactivity was compared in several species, including cows, humans, and rats, Eriksson et al63 demonstrated that high levels of desmin were correlated with the morphologically well-differentiated Purkinje fibers of hoofed animals and that low levels of desmin were correlated with the morphologically poorly differentiated ventricular conduction system of the rat. This notion is in line with a study of rat heart development by Ya et al,64 who concluded that the expression of smooth muscle proteins and desmin is a temporary parameter for the process of myofibrillar organization in the developing cardiomyocyte. They observed an only slightly higher expression of desmin in the prenatal and postnatal ventricular conduction system.Finally, it has been reported that 1 kb of the human desmin promoter specifically drives transgene expression in the cardiac conduction system, as judged from in toto X-galactosidase staining of mouse embryos at 8 days and at later stages.97 The endogenous gene is expressed in the entire early embryonic heart.98 In this early stage, expert transmission electron microscopy305361 has not allowed the unambiguous recognition of the conduction system. Moreover, the presence of expression of the transgene in the conduction system requires analysis of histological sections rather than of intact hearts on in toto staining.NeurofilamentThree types of neurofilament have been described in mammals with molecular weights of ≈70, ≈150, and ≈200 kD, dubbed NF-L, M, and H, respectively.99 Immunoreactivity for the L and M subunits was found in all parts of the adult rabbit conduction system.100 Data have been substantiated at the mRNA level.101 Neurofilament mRNA can be detected from 9.5 days of development onward, and the protein is detectable slightly later at the sinoatrial junction and at both sides of the wall of the AVC of the rabbit heart, where it colocalizes with desmin.102 It may be of interest to analyze neurofilament expression in conjunction with connexin isoform expression (to delineate the nodal areas567273 ) during rabbit cardiac development. This would permit the distinction of the nodal areas from the specialized fast-conducting atrial tracts that could also be positive for neurofilament100103104 [see “The Atrial (Internodal) Conduction System” below].RecapitulationIn an uncomplicated view, the phenotype of the nodal myocytes could be dubbed “embryonic” because of their electrical (embryonic conduction velocities and action potentials) and contractile (embryonic isoforms and poor sarcomeric organization) characteristics. Nevertheless, it seems too simplistic to consider the nodes as mere remnants of the embryonic myocardium, since they have become physiologically highly specialized. Elevated levels of the calcium-release channel/type-1 inositol trisphosphate receptor,105 γ-enolase,106 α2 and α3 isoforms of the sodium pump (Na+,K+-ATPase),107 G protein α-subunit,108 and the AT2 receptor subtype109 have been reported in the adult node and/or ventricular conduction system compared with the working myocardium. It is to be expected that analyses of the developmental patterns of expression of this type of proteins will provide insight in the maturation of the nodes in the near future. Such studies may shed new light on a number of intriguing questions regarding the development of the nodes. For example, how do cells of the primary myocardium escape differentiation into working myocardium of atrium and ventricle, and what causes them to differentiate into nodal direction? How do they sort out positionally to form the intricate nodal structures of the formed heart, and what type of interactions with surrounding myocardial and nonmyocardial cells are involved?Development of the Ventricular Conduction SystemIn the formed mammalian heart, the ventricular conduction system comprises the AVB, left and right bundle branches, and a PPN, which extends into the periarterial Purkinje fibers in birds only132 (Fig 5a). There exists a great degree of interspecies variability in the type of Purkinje cells, which are clearly distinct from the working myocardium in birds and hoofed animals, less clear in humans and dogs, and almost indistinguishable, or perhaps even absent, from the working myocardium in rodents.2110 Also, there exists within a species considerable diversity that is related to the position of the cells in the conduction system, with those at the periphery (transitional cells) being almost indistinguishable from the working myocardium.59111 In fetal and neonatal human hearts, an additional right atrioventricular ring bundle has been described,112 whereas in the embryonic human heart a septal branch and a retroaortic root branch have also been described.113 Finally, in the adult avian heart, the entire system is present.32111 How does this system develop, how does it fit in the model of the segmented heart, and what is its origin?Morphological DevelopmentA closer look at Fig 5a reveals two component parts in the ventricular conduction system. The first part is a “drape-like” part that is positioned on top and astride the ventricular crest (AVB and bundle branches), extending at the luminal side of the ventricular myocardium. It penetrates into the compact myocardium. This part brings the depolarizing impulse to the ventricles. Its position in the developing tubular heart can easily be envisioned (Fig 5b). The second part of the ventricular conduction system (Fig 5a) surrounds the subaortic outlet of the ventricle and the right atrioventricular junction just above the atrioventricular annulus. It is a bended oval ring, which, depending on the optic angle, will be perceived as a figure-8–shaped ring. This ring of myocardium has been demonstrated in the early fetal human heart and could be traced back to the myocardium surrounding the primary interventricular foramen in the 5-week embryonic heart113 (Fig 5b).To understand the remodeling of the primary interventricular foramen, one should appreciate that the term “interventricular foramen,” used in many textbooks, is confusing, because it is not interventricular only, as will be pointed out below. The ventricular compartments develop from the primary heart tube by the formation of trabecular pouches114115 (Fig 2). The ventricular septum develops by apposition of ventricular myocytes at the outer side, leaving a foramen, dubbed the primary interventricular foramen, between the inner curvature and the top of the ventricular septum. With diastole, both ventricles are filled (the right ventricle via the primary interventricular foramen); with systole, both ventricles are emptied (the left one via the primary interventricular foramen); and so the “crossing” flows of blood are separated in time. Thus, the essence of the position of the primary interventricular foramen is that it demarcates both the inlet of the right ventricle and the outlet of the left ventricle. As will be clear from Fig 5a, this is still the position in the formed heart. With septation, the primary interventricular foramen becomes divided by extension and fusion of the endocardial cushions. By this process, the right atrioventricular junction becomes physically separated from the left ventricular outlet.The developmental fate of the myocardium surrounding the primary interventricular foramen has been followed in human cardiac development113116117 (Fig 5c to 5e) on the basis of the expression of an epitope, dubbed GlN2 because it reacts with an antibody raised against an extract from the