Developmental Biology of the Heart

Overview of Cardiac Developmental Anatomy

This chapter is a review of embryonic and fetal cardiac anatomy, including the cell types that contribute to the developing heart, and genetic regulation of heart development. We will follow the heart from its stages as a linear tube with inflow and outflow segments, through cardiac looping and septation into a four-chambered heart with separate pulmonary and systemic circulations. Understanding these processes is of interest to clinical neonatologists caring for neonates and infants with normal and abnormal cardiac physiology.

Heart development begins at gastrulation as a field of cells; cardiac mesoderm ( Fig. 45.1A ) is specified and moves ventrally and midline within the embryo, fusing into a tube. This occurs in the human embryo by 3 weeks, post-fertilization, or approximately week 5 after the last menstrual period ( Fig. 45.1B ). At the posterior/dorsal end of the tube is the venous pole, or sinus venosus, connecting to the systemic and placental venous system. At the anterior/ventral end of the tube is the arterial pole, or outflow tract, which connects to the developing aorta. The heart tube itself is divided into an atrial segment, adjacent to the venous pole, and atrioventricular (AV) canal, and a ventricular segment adjacent to the arterial pole. Each of these segments is histologically and functionally distinct from the beginning—atrial and ventricular myocytes have distinct characteristics even when isolated from the primitive mesoderm. By the time the heart tube is formed, differentiating cardiomyocytes have generated functional contractile units mature enough to begin spontaneous contractions. These are initially peristaltic in nature, moving blood in one direction from the venous inlet to the arterial outlet. This is initially clear fluid, but red blood cells begin to enter the circulation from blood islands in the yolk sac soon after the heart starts to beat. For the first 6 weeks, the yolk sac remains the exclusive source of hematopoietic cells, until the liver (and to a lesser extent spleen) takes over. The bone marrow gradually becomes populated with hematopoietic cells beginning in the second trimester of pregnancy, and by birth is the major source of blood cells. Development of a circulatory system early is critical to maintaining nutrition and oxygen delivery to developing tissues, which are at this point beyond the ability to depend on simple diffusion of nutrients.

From the primitive heart tube state, the heart undergoes significant growth and morphologic alterations. The symmetry of the heart tube is lost and well-defined left and right structures acquire their functions as the heart undergoes a dramatic change in shape, via a process known as cardiac looping. This occurs in the human embryo during weeks 4 to 5 of gestation (see Fig. 45.1B ). From this stage, the heart realigns and septates the inflow and outflow segments, septates the atria and ventricles, and forms the atrioventricular and semilunar valves in processes that will be detailed in this chapter. The “final product” will be a mature heart, formed by gestational week 7 (or 9 weeks after the last menstrual period; Fig. 45.1B ) with the following structures:

• 1.

  A venous pole or sinus venosus (red) that is now connected to systemic veins from the upper body (superior vena cava), lower body (inferior vena cava [IVC]), liver, and coronary circulation, all of which pass into the right atrium (RA) .

• 2.

  A separate left atrium (LA) , connected to the venous system separately by ingrowth of pulmonary veins from the lungs.

• 3.

  An atrioventricular canal that begins as a single unrestricted opening, and septates into two atrioventricular valves. The tricuspid valve opens from the right atrium into the right ventricle, and the mitral valve opens from the left atrium to the left ventricle.

• 4.

  A primitive ventricle that has expanded and septated into two distinct and separate chambers, a right and left ventricle . By processes known as compaction and trabeculation, the working myocardium of each ventricle becomes highly organized and adapts to the unique requirement of a pulmonary (RV) or systemic ventricle (LV).

• 5.

  An arterial pole or bulbus cordis that forms the two separate outflow tracts of the heart. The distal part of the bulbus cordis is the arterial trunk , which undergoes septation in a spiral fashion creating an anterior pulmonary artery connecting to the lungs and a posterior aorta connecting to the body. The proximal part of the bulbus cordis is the conus , which is retained by the right ventricle. At the junction between conus and the pulmonary artery, the pulmonary valve forms. The aortic valve forms at the junction between the left ventricle and the aorta.

• 6.

  Paired dorsal aortae connected to the aortic sac that have extensively remodeled into a single leftward aortic arch and descending aorta . This connects proximally to the coronary arteries , the head and neck arteries, and to a temporary structure important in fetal life, the ductus arteriosus .

• 7.

  Networks of coronary arteries that have formed within the myocardium of the heart. These connect to the aorta as the right and left coronary arteries.

• 8.

  Networks of myocytes that have differentiated into the conduction system of the heart, including the sinoatrial node, the atrioventricular node, and the His-Purkinje system of the ventricles.

Cell Types Within the Heart and Their Origins

The mature heart has three cell layers: the endocardium, a single cell thickness epithelial lining; the myocardium, made up of myocytes that perform the contractile work of the heart; and the epicardium, a single cell thickness epithelium covering the external surface of the heart. Beyond this, the heart resides within a pericardial sac, similar in composition and function to the pleura covering the lungs.

The heart proper has its embryonic origin from a field of lateral plate mesodermal cells referred to as the cardiogenic mesoderm ( Fig. 45.1A and B ; orange / red ). The primitive heart tube arises from the primary heart field ( Fig. 45.1A and B , orange ) and has two layers, an endocardium and a myocardium, both arising from cardiac mesoderm. A subset of cells from the endocardium undergoes an epithelial-to-mesenchymal transition to form the endocardial cushions. These cells will be vital to proper valve development and to complete septation of the atria and ventricles. The myocardium primarily remains muscle, but a subpopulation of these cells also differentiate into Purkinje fibers of the conduction system.

The outflow tracts of the heart are arguably the most complex genetically and morphologically, and thus most prone to developmental defects. The secondary heart field is a population of cardiac mesoderm adjacent to the original heart field that migrates into the heart during looping ( Fig. 45.1A and B , blue ). The secondary heart field contributes significantly to the right ventricle and outflow region of the heart, to the atrial and ventricular septa, and to parts of the atria.

Neural crest cells are an important population that also migrate into the developing outflow tract, interacting with the secondary heart field myocardium ( Fig. 45.1A and B , green ). Neural cells originate from the ectoderm in the anterior rhombencephalon and migrate as a sheet through the pharyngeal region and into the aortic arches, truncus, and proximal conus. Here they interact with endocardial cushion cells to septate the great arteries and close the conal septum. These neural crest cells are also important to the development of the nearby parathyroid, thyroid, and thymus glands. They also innervate the heart and form much of the smooth muscle of the proximal aorta.

Another external population of cells that make important contributions to the mature heart is a cluster of cells dorsal and inferior to the heart tube known as the proepicardial organ ( Fig. 45.1A and B , purple ). The origin of these cells is a subject of debate; one leading theory is that they are derived from liver primordium. These cells expand as an epithelial sheet covering the surface of the heart to form the epicardium. From the epicardium, subgroups of cells delaminate and migrate into the myocardium beneath in a process known as epithelial to mesenchymal transformation. These cells differentiate into vascular smooth muscle, vascular endothelial cells of the coronary arteries, and cardiac fibroblasts, which make up a sizable population of cells residing within the myocardium, between myofibers. A recent study using multiple lineage markers in tandem showed that only about 4% of the coronary endothelium comes from the pro-epicardium, while the contribution to fibroblast and smooth muscle population is much higher. This study identified a population of circulating endothelial progenitors as an important source of cardiac endothelium.

Thus in addition to cardiomyocytes arising from at least two populations of mesoderm, the heart is composed of cells from epithelial and neural crest origins that migrate in with spatial and temporal precision during cardiac development. The next several sections will highlight the steps of this process.

Gastrulation and the Cardiac Crescent

Much of what is known about the early stages of embryonic development comes from studies of avian, zebrafish, and mouse models extended, with some acknowledged gaps, to human development. In its earliest stages, the embryo exists as a bilayer disc of two epithelial sheets of cells suspended between two fluid-filled cavities, the yolk sac and the amniotic cavity ( Fig. 45.2A ). The ventral layer (facing the yolk sac) is the hypoblast, which will eventually be relegated to extraembryonic structures. The dorsal layer (facing the amniotic cavity) is the epiblast, which will form all three embryonic germ layers: the ectoderm (nervous system and skin), mesoderm (heart, skeleton, muscle, and connective tissue), and endoderm (gut). Mesoderm and embryonic endoderm separate from the primitive ectoderm by a process known as gastrulation. Gastrulation begins as a groove that forms in the epiblast starting at the tail end (caudal) and gradually extending cranially to an endpoint known as the prechordal plate ( Fig. 45.2A ). The groove is known as the primitive streak, and its leading edge is the primitive node. Along the primitive streak, epiblast cells delaminate from the epithelial sheet and invade the potential space between the epiblast and hypoblast, forming a crescent of cells cranial to the prechordal plate and extending along both sides ( Fig. 45.2B ). Genetic and fate mapping studies also show that the relative position of cells during migration is key to their ultimate position within the heart. Specifically, the apex of the crescent is formed from cells that have migrated through the primitive streak closest to the primitive node, and these are precursors of the outflow tract myocardium. The cells on either side of this region have migrated through the mid-portion of the streak and are ventricular myocyte precursors. The most lateral and caudal cells are those which have migrated through the most posterior part of the streak and will become atrial myocytes ( Fig. 45.2B ). Finally, the secondary heart field mesoderm lies medial and extends along the entire crescent such that it is continuous with both the outflow and inflow segments of the crescent ( Fig. 45.2B , green). Fate-mapping studies using live-cell tracking and time-lapse imaging demonstrate that these cells move as a cohort (tissue motion) rather than individually migrating; this is a way to maintain cells’ relative positions within a tissue, and eventually, an organ.

Genetic Control of Gastrulation

The earliest known marker of the cardiomyocyte lineage is the transcription factor Mesp1, expressed in the mesoderm at the onset of gastrulation. Mesp1 acts as a switch between pluripotency (phenotypically linked to mesenchymal, migratory, and proliferative characteristics) and differentiation.

At the primitive node, growth factors of the wingless integrated or Wnt family are expressed which block differentiation and promote proliferation and migration. Surrounding structures, including the neural tube and endoderm, secrete inhibitory factors forming the boundaries of the crescent-shaped heart field.

Thus as the migrating cells make their way into the cardiac crescent, they exit the region of Wnt expression and enter a field of active Wnt inhibition. The cardiac mesoderm then begins to differentiate into cardiomyocytes under the control of a different class of growth factors, bone morphogenetic proteins (BMP). Early markers of the differentiated cardiomyocyte phenotype include transcription factors Islet 1, TBX5, GATA4, and Nkx 2.5. The mechanisms for the shifts in gene expression are complex and involve many epigenetic factors including modification of chromatin structure in differentiating cells and enhancer and repressor regulatory elements. Factors such as GATA4 function both to modify chromatin structure, for example, cause “unwinding” of specific segments of DNA rendering it accessible to transcription, and to bind to certain gene promoters to enhance transcription directly. Furthermore, Gata4 recruits TBX5 to muscle-specific gene promoters in the primary heart field; in mice TBX5 deletion causes abnormal development of the left ventricle. In humans, TBX5 haploinsufficiency causes atrial septal defect (ASD), conduction defects and limb anomalies, but can also be associated with hypoplastic left heart syndrome. Similarly, both NKX 2.5 and Islet1 function by binding to regulatory elements of cardiac-specific genes, including chromatic structure and transcription enhancer elements; there is additional complexity in this system from the interdependency and to some extent redundancy of GATA4, NKZ2.5, and TBX5.

The basic left-right asymmetry of the embryo is set during gastrulation by concentration gradients of the factors sonic hedgehog (shh) and fibroblast growth factor 8 (see Fig. 45.2C ). This gradient is created by ciliary motion at the primitive node and causes a cascade of downstream genes to be activated, including nodal, lefty, and pitx-2. All of these are markers of left-sidedness, whereas as far as currently understood right-sidedness is specified entirely by the absence of these factors. When the strict left/right gradient of gene expression is altered, the result is abnormalities of left/right structures in the heart, lungs, and/or gut known as heterotaxy (see section below and Fig. 45.4 ).

Looping and Laterality of the Heart Tube

When initially formed the cardiac crescent is a symmetric structure, and it next fuses into an initially symmetric heart tube. This primitive heart tube forms as the cardiac crescent moves ventrally and medially, such that the two arms of the crescent fuse into a tube ( Fig. 45.3A ). The developing foregut dorsally and the neural tube cranially help to push the heart tube into its new position. As fusion of the heart fields occurs, the heart begins to beat. In a human fetus, this happens at approximately 23 days’ gestation or early week 6 after the last menstrual period.

The heart tube next begins to undergo a dramatic change in shape known as looping. Here the heart tube loses its symmetry, and distinct left and right morphology can be identified ( Fig. 45.3B ). Morphologically, cardiac looping involves some degree of differential growth, with a higher proliferation of myocytes along the outer curvature than the inner. The primary contribution to the morphologic change during looping, however, is a substantial migration of cells from the developing secondary heart field. Of note, this migration of secondary heart field cells occurs both at the arterial and venous poles of the heart. All of this serves to elongate the outflow tract and enlarge the ventricular segment. Recent detailed lineage tracing and studies in chick heart tubes confirm that the entire right ventricle arises from the secondary rather than the primary heart field. The atria and sinus venosus also incorporate secondary heart field cells, and this portion of the heart tube is shifted cranially from its original position. Overall, the heart tube increases fivefold in length during looping. In addition to cell migration and proliferation, there are mechanical forces literally pulling, twisting, and realigning structures of the primitive heart tube. This is thought to involve the cytoskeleton, including non-muscle myosin, the motor protein dynein, microtubules, and non-muscle actin bundles.