In the primary embryonic heart tube, the atrioventricular canal with the endocardial cushions completely connects to the primitive left sided ventricle. The OFT, initially situated entirely above the primitive RV, connects to the unseptated aortic sac that still needs to be divided into a pulmonary and aortic orifice leading to the great arteries. As mentioned above, during development the heart tube shows a dextral looping leading to a marked almost circular groove which is referred to as the inner curvature. The inner curvature is reflected internally by the primary fold or ring positioned between the primitive LV and the RV, which is still expanding due to the material addition of the SHF (Fig. 1.1). Both the atrioventricular canal myocardium and the nontrabeculated component of the OFT are lined by endocardial cushions. During remodelling and septation of the OFT, the aorta becomes connected to the LV. The long held idea was that rotation of the endocardial cushions was instrumental together with additional shortening of the subaortic OFT myocardium [26]. The mechanisms underlying rotation as well as the inferred apoptosis (programmed cell death) in the OFT have always remained hypothetical. We discovered an asymmetric and late contribution of Nkx2.5 expressing primitive mesenchyme (see Ref. [27]) on top of addition of smooth muscle cells preferentially to the pulmonary side of the OFT [28]. Analyzing this in 3D reconstructions we devised a new hypothesis in which rotational movement must be substituted for an anterior push of the subpulmonary myocardium resulting in lengthening of the RV OFT (Fig. 1.3 and Supplemental Video 1.1). This mechanism eliminates the requirement of marked subaortic apoptosis to understand the relative low position of the aortic orifice wedged between the tricuspid and the mitral orifice.

The dextral looping process and the relocation of the pulmonary orifice includes a marked relocation of the atrioventricular (AV) canal allowing the future tricuspid orifice to channel into the RV inflow tract compartment. Several concepts for this remodelling process have been proposed. Initially, the right side of the AV canal with the putative tricuspid orifice is positioned to the left of the primary fold (Fig. 1.1). The originally very small RV inflow tract enlarges and becomes positioned to the right of this fold. The exact mechanism in which this is achieved still remains elusive but concepts will be described in the section on ventricular septation.

The primary heart tube consists of an endocardial inner surface, a thick basement membrane, referred to as cardiac jelly, and an outer myocardial layer. During looping and SHF addition, the cardiac jelly persists in two endocardial cushion areas situated in the AV canal and in the OFT. Both cushion areas play a role in cardiac valve formation as well as in septation.

The remaining cardiac tube forming the future LV and RV is a two layered thin structure that borders on the outside to the coelomic cavity. Subsequently, two essential processes take place. First, the future outer compact myocardial layer and an inner trabecular layer become distinct, and second, progressive myocardial compaction needs to occur. The mechanism underlying trabecular formation, linked to AV valve development, has recently been described [29]. The system of trabeculations has an important mechanical function in the heart tube [30] maintaining ventricular wall strength. The trabecular sinuses also serve as a reservoir for blood. Before the complete development of the RV there is little difference in the trabecular architecture of the RV and LV [31] (Fig. 1.4). The compact layer of the myocardium still has to increase in thickness and interaction with the epicardium is an essential component of this process.

Most of the epicardium is derived from the so-called pro-epicardial organ (vPEO) positioned at the venous pole of the heart [32]. Recently we have shown that a second epicardial organ (aPEO) can be distinguished at the OFT (Fig. 1.2). Epicardial cells migrate from both sources over the heart (vPEO derived) and great arteries (aPEO derived). The anterior surface of the RV is the last to be covered in this process [33]. The epicardium becomes relatively dormant in adult life. However, the role of the epicardium in cardiac development has received the attention of many research groups because myocardial injury is associated with adult reactivation, which may play a role in repair after myocardial infarction, Several recent reviews cover this area [32, 34–36] containing the information on the underlying primary research.

The data that reflect specifically on the development and morphology of the RV will be presented here. The epicardium derived from the vPEO is referred to as cardiac epicardium (cEP) as it covers only the myocardium up to the ventriculo-arterial junction. After this covering a process of epithelial-mesenchymal-transition (EMT) leads to a population of epicardium derived cells (EPDCs) that move into the subepicardial space and subsequently migrate into the myocardium. Many genes and molecular pathways are involved in this process [37]. Having entered the myocardium, EPDCs will differentiate into the smooth muscle cells of the coronary arteries and into intracardiac fibroblasts. It cannot be excluded that these populations are already programmed to their fate while still being on the surface [38] which might, therefore, harbour a heterogeneous epicardial population. When epicardial outgrowth [39], the process of EMT or migration are disturbed [32], normal compaction of the ventricular myocardium will not take place leading in the most severe cases to a very thin compact myocardium or embryonic death. This has been described in many animal models based on mutated genes influencing the epicardium or the ensuing cross-talk between epicardium and myocardium (an example is shown in Fig. 1.4 for a TGFβ mutant mouse). This important population for myocardial differentiation shows a temporo-spatial difference in the contribution to the RV and LV wall (unpublished data). In summary (1) the EPDC migration into the RV precedes slightly the migration into the LV wall, (2) eventually the LV receives more EPDCs and (3) there is a difference in anterior to posterior deposition of EPDCs in the RV wall. The RV receives relatively more EPDCs than the LV, and in both ventricles the posterior wall holds more EPDCs than the anterior wall. Whether this observation is relevant for adult RV and LV function is not known but it is tempting to relate it to non-compaction cardiomyopathies and the difference between RV and LV types. In several forms of congenital heart disease, the posteriorly situated inflow compartment is mostly affected, as is described for pulmonary atresia with intact ventricular septum resulting in a bi- or unipartite ventricle.

The EPDCs that differentiate into smooth muscle cells start to cover the main arterial stems that have grown into the aorta at specific sites of the aortic wall [40, 41]. These initial observations in avian embryos are now confirmed in mouse studies [42]. The reason that normally the coronary vessels do not penetrate the pulmonary wall is postulated to relate to the specific myocardial origin of the subpulmonary OFT.

Disturbed epicardial contribution can lead to diminished coronary vascularization of the myocardium as well as absent or pin-point coronary arterial orifices [41, 43]. The relevance for specific RV pathology might relate to the incompletely understood preference of ventriculo-coronary-arterial communications (VCACs) or fistulae in case of pulmonary atresia without ventricular septal defect (VSD) in the hypoplastic RV [7, 8]. A similar coronary arterial pathology is not seen in the hypoplastic left heart [44]. It is unclear whether myocardial architecture dictates distribution of the microvascular coronary capillaries or vice versa. It is obvious that the normal LV has a more regular patterning of capillaries compared to the RV [45].

The RV morphology is clearly different from the LV based on a number of structural characteristics of the RV cavity. The entrance into the RV from the right atrium is via the tricuspid orifice which is encircled by a fibrous annulus composed of fine collagen. The tricuspid valve consists of three valve leaflets: the posterior, the anterior and the septal leaflet. These leaflets are attached by chordae tendineae to the papillary muscles including the septal and moderator band. In general the RV is divided into three parts: the tripartite division [46]. Following the flow, these consist of (1) a posteriorly located inlet segment. Subsequently, crossing the septal to moderator band continuity (Fig. 1.5) (2) the trabecular component reaching up to the apex. The latter apical compartment is lined by relatively coarse trabeculations that distinguish the RV from the finely trabeculated LV. The last compartment is (3) the relatively smooth walled OFT leading to the pulmonary orifice and pulmonary trunk. The pulmonary orifice harbours three semilunar valve leaflets. The anchorage of this orifice into the OFT myocardium differs from the way into which the aortic orifice is embedded into the LV [47–49].