Chamber differentiation occurs during further rightward looping of the heart tube, which results in positioning of the ventricles and the outflow tract of the heart in an anterior/ventral position, and of the atria in a dorsal/posterior position (Fig. 1.3). Transcriptional regulators (Nkx2-5, Tbx1, Tbx2, Tbx3, Tbx5, GATA4, Irx3 along with many others) and signaling pathways (including Notch, WNT, Bone Morphogenetic Protein [BMP], and Retinoic Acid) control chamber differentiation and formation of septal structures, the valves, and the great arteries [2, 3]. Electrical activity occurs early during development of the heart in conjunction with further differentiation of the simple heart tube into a four-chambered structure [4].

There has been much controversy with regard to the origin of the specialized myocardial tissue that leads to the development and expression of the conduction system. Current understanding suggests that cardiac myocytes, rather than neural crest cells, for example, are the progenitors of specialized conduction tissue. These findings were primarily supported by retroviral reporter gene transfection lineage studies [5–9]. The exact factors dictating this differentiation and development, however, remain to be elucidated, but it appears that neuregulin plays a crucial role in this differentiation process [10–16].

In a brief review in 1976, Wenink and colleagues proposed that there were four rings of specialized tissue in the embryo that could be distinguished from the surrounding myocardium once looping of the heart had occurred [17]. These four rings (Fig. 1.4) were thought to mark transitional zones of the heart and included: the sinoatrial ring, between the sinus venosus segment and the primitive atrium; the atrioventricular ring, between the primitive atrium and primitive left ventricle; the primary ring or fold, that separates the primitive left ventricle from the primitive right ventricle; and the ventriculoarterial ring, at the junction of the primitive right ventricle and the truncus or putative outflow tract of the heart [1] (Fig. 1.3). It is thought that during completion of looping of the primitive heart tube, these four rings come together in the inner curvature of the heart and with further differentiation; part of this tissue loses its specialized character. What remain of the rings become the definitive elements of the mature conduction system. According to this theory, the sinoatrial ring contributes to the formation of the sinoatrial node; both the sinoatrial ring and the atrioventricular ring contribute to the atrioventricular node. The primary ring gives rise to the His bundle and bundle branches while the ventriculoarterial ring regresses almost entirely.

Studies in the 1990s used the expression pattern of a neurofilament-like protein as a marker for the developing conduction system. The presence of neurofilament-like protein was used to demonstrate a ring at the sinoatrial and atrioventricular junctions, and in ventricular components of the developing conduction system, which were distributed in the ventricular subendocardium and connected to the atrioventricular ring [18–22]. In contrast to the theory that local cells undergo specialized differentiation, other studies suggest that conduction tissue cells (of the rabbit heart, for example) may originate from a population of neural crest-derived cells migrating from the branchial arches into the developing heart [20].

As these conflicting theories continued to be investigated, several immunohistochemical and molecular markers for cardiac conduction system development were used to support the hypothesis that conduction system cells differentiate from local cells. Even though none of the immunohistochemical markers are truly specific for labeling specialized conduction system cells, supportive evidence seemed to favor the “four ring theory.” Using a monoclonal antibody to HNK1 antigen, for example, investigators demonstrated findings consistent with the notion that rings of conduction system tissue exist and undergo further differentiation (Fig. 1.5). HNK1 is predominantly expressed in the developing sinoatrial and atrioventricular junction of the conduction system, and the expression pattern seems to correspond with the rings described early on by Wenink. In human embryos, antibodies to HNK1 antigen stains the sinoatrial node, the internodal myocardium in the right atrium, the right atrioventricular ring with a future posterior and anterior atrioventricular node, a retroaortic ring, the His bundle, and the bundle branches. It appears that the myocardium surrounding the primitive pulmonary veins also demonstrates transient staining of HNK1 [23].

Podoplanin is a 43-kd, mucin-type transmembrane glycoprotein that is found outside the heart in several organs and tissues [24–32], such as osteoblasts, the nervous system, epithelia of lung, eye, esophagus, and intestine, mesothelium of the visceral peritoneum; the podocytes of the kidney; and lymphatic endothelium [33–36]. It is thought that podoplanin expression in the developing heart is a marker for the developing sinus venosus myocardium, supporting its development from the posterior heart field. Podoplanin is expressed in the areas that are in close contact with the sinoatrial nodal myocardium and in the underlying mesenchyme adjacent to the cardinal veins. It appears that podoplanin-positive mesenchyme differentiates into myocardium that stains negative for Nkx2.5. During cardiac development, podoplanin is expressed in myocardium along bilateral cardinal veins and in both the right-and left-sided sinoatrial nodes. This expression is maintained on the right as part of the right sinus node and right-sided venous valves, at the base of the atrial septum, the posterior atrioventricular canal, the atrioventricular nodal region, and the His-Purkinje system; it is opposed by the expression of Nkx2.5. Also, during later developmental stages, podoplanin is expressed in the pulmonary veins. In podoplanin negative mice, myocardial components around pulmonary veins are reduced and there is underdevelopment of the atrial septum [28, 37]. It appears that podoplanin plays a critical role in myocardial tissue associated with the sinus node and that abnormal epithelial-to-mesenchymal transformation of the coelomic epithelium due to up-regulated E-cadherin and down-regulated RhoA impose abnormalities in the formation of cells that form the sinus venosus [28].

Even though the development of anatomical structures supporting the specialized conduction system offers insight into genesis of the conduction system, functionally, the development of impulse generation and propagation remains to be fully understood. In the mature heart, the sinoatrial node is the primary pacemaker of the heart, and impulse propagation occurs through the atrioventricular node and specialized His-Purkinje system. Impulse propagation, itself, can be further divided into fast, as in the His-Purkinje system, and slow as seen in myocardial tissue, and slower yet, as seen in the atrioventricular node [6]. Different animals reveal complex variations in the organizational and functional components of the conduction system [7, 8, 38–40]. For example, the study of the chick embryo allowed understanding of a pacing generator around 25–35 h of development in the posterior most part of the tubular heart [41]. Similarly, pacing activity is noted around 7.5 days and 21 days in mice and humans, respectively [42]. At this time, the heart consists of a simple heart tube and the initial contractions are slow and rhythmic [43], but establish unidirectional flow and posterior to anterior polarity [44–48]. These peristaltic contractions can be recorded, inscribing a sinusoidal ECG [49].

Furthermore, there appear to be transient expression of key transgenic markers, timed chronologically, that determine developmental fate of myocardial cells. For example, the heart tubes in zebrafish, chicks, and mice appear to have retinoic acid-sensitive markers along the heart tube that dictate formation of atrial tissue [50]. Retinoic acid appears to control atrial-specific gene expression and exclusion of retinoic acid from ventricular tissue precursors seems essential for correct specification of the ventricular muscle development. In addition, transmembrane hyperpolarization-activated cyclic nucleotide-gated family of ion channel subunits plays a key role in impulse generation supporting pacemaker activity, both in the embryo and the adult human heart [49, 51–53]. Other genes also play a role in impulse generation as can be seen in studies that show that knock-out of the NaCa exchanger gene causes mortality due to inhibition of pacing function in the tubular heart [54]. Along with further differentiation, the developing atrial and ventricular myocardial cells acquire high conductance gap junctions that can then support rapid transmission of an electrical impulse by rapid proliferation and up-regulation of genes. These working myocardial cells have increased mitochondria and increased sarcomere components.

In contrast to rapidly proliferating myocardial tissue, cells in the atrioventricular canal area retain their slow proliferation rates, and also retain their “embryonic-like” mode of conduction, which is much slower [51, 55, 56]. In association with chamber formation, slow wave propagation producing peristaltic contractions are replaced by rapid depolarizations (and contractions) of cells of the atrium and ventricles, inscribing an ECG that resembles the one of the mature heart. These changes seem to occur in parallel with anatomical looping of the heat tube. Differential expression in conduction velocities of the conduction system components in the mature heart accompanies looping. These structural changes parallel a delay in conduction time in the mature atrioventricular node [57–59]. In fact, in the adult myocardium, the impulse proceeds from the sinus node to the crux of the heart at 0.1–1.0 m/s and is slowed at the atrioventricular node to 0.01–0.05 m/s, increasing its velocity to 2–4 m/s in the His-Purkinje system, with a decrease to 0.3–1.0 m/s in the ventricular myocardium [4]. Thus, sequential contraction of the atrial and ventricular chambers in higher species is dependent on the specific functional development of atrioventricular delay [5, 6, 16, 43, 60, 61]. This delay can be seen at 42 h of development in the chick and at 8 and 25 days in the mouse and human [6, 7], respectively. Furthermore, in the looping heart, there are two other areas of relatively slow conduction: the sinoatrial area and the outflow tract area. This slow conduction is associated with the expression of connexin 45, which is characterized by high voltage sensitivity and low permeability [62–65]. Knock-out mice of the connexin 45 gene result in death from heart block at looped, tubular stages of heart development [66, 67].

The His-Purkinje system is the last component of the conduction system to differentiate. In mammals, differentiation of the His-Purkinje system is quite advanced, resulting in markedly efficient and coordinated myocardial activation and associated myocardial contraction [5, 8, 68]. Retroviral lineage studies suggest that central and distally located components of the His-Purkinje system differentiate separately, but then link together during development [7]. In 1999 and 2000, differentiation of the His-Purkinje system in ventricular myocytes was found, in the chick embryo, to be induced by endothelin-1, secreted from adjacent coronary arterioles [69, 70]. This particular finding was not noted in the mouse, however. Similarly, some in vitro evidence suggested that neuregulin-1 played a role [15, 71, 72] in His-Purkinje development. In addition, cellular studies observed that there is a switch in activation sequence in the developing heart. The emergence of the mature His-Purkinje system in the developing chicken embryo had been studied using anti-polysialylated neural cell adhesion molecule (PSA-NCAM) and the HNK1 antibody against a sulfated carbohydrate epitope (antigen). The appearance of the mature form of the His-Purkinje system coincided with the onset of the mature electrophysiological patterns of ventricular activation. These data suggested that, at the completion of ventricular septation, the His-Purkinje system undergoes critical structural and functional transitions that impacted the global pattern of impulse conduction and contraction of the developing four-chambered heart [73, 74]. Using cardiac conduction system-lacZ line of reporter mice, several investigators tested the ability of endocardial-derived and secreted (paracrine) factors to convert contractile cardiomyocytes into conduction system cells. It appeared that neuregulin-1, a growth and differentiation factor essential for ventricular trabeculation was sufficient to induce ectopic expression of a lacZ conduction marker. In the mouse model, this inductive effect of neuregulin-1 was restricted to a window of sensitivity between 8.5 and 10.5 days fertilization. Thus, it appeared that endocardial-derived neuregulins may be responsible for inducing murine embryonic cardiomyocytes to differentiate into cells of the conduction system [15]. In a similar manner, Gassanov et al. described differentiation of atrial-derived cardiomyocytes to a pacemaker-like phenotype induced by endothelin-1, but not associated with neuregulin [72].