Myocardium and Development

Cardiac Looping

Structure underlies and ultimately determines function. Our understanding of the identity of the cells that populate and function in the heart, and their origins, has changed radically in the past 15 to 20 years. The generally accepted concept of an early heart tube containing all the necessary precursors of what will become a mature heart has been shattered by new technologies that allow the detailed dissection of cell lineages, sites of proliferation, and migration. Studies from the worm, fly, chicken, and mouse have enriched our understanding of human heart development as well, and we now know that many of the precursors for the various cell types in a mature heart are added to the primary heart tube at both the venous and arterial poles.

Molecular genetics has provided the tools to carry out detailed cell lineage tracings, showing clearly where the different precursors for the various components of the heart arise, how quickly they migrate, proliferate, and finally, when they express the specific proteins that serve as markers for the mature cell type. With the important exception of neural crest cells, which are derived from the neuroectoderm and contribute cells to the outflow tract and other, critical cardiac structures such as the valves and connective tissues as well as other organ systems, current data confirm that the heart is largely derived from the mesoderm. By 15 days a primitive streak containing cells that will migrate anteriorly is discernable ( Fig. 5.1 ). By 3 weeks, the human embryo contains a clearly recognizable structure populated by cardiac precursors located in the mesoderm, which is termed the cardiac crescent (see Fig. 5.1 ). The splanchnopleuric layer, which faces the endoderm, will give rise to the basic cardiac structures. The bilateral-located precursors fuse at the midline to form the cardiac crescent, and chick embryonic cells isolated from the same approximate developmental stage can be detected expressing proteins, such as cardiac-specific transcription factors, and a few sarcomeric proteins.

In human embryonic development, the cardiogenic region and primitive blood vessels of the embryo is present ~18 days postovulation (CS 7). Blood flow through the endocardial tubes and fusion of the two tubes into a midline structure is detectable at 20 days, with five recognizable segments of the heart tube (i.e., the truncus arteriosus, bulbus cordis, primitive ventricle, primitive atrium, and sinus venosus) at 22 days. Contraction results in the propulsion of blood from the sinus venosus to the truncus arteriosus. Looping of the primitive heart tube commences 23 days postovulation (CS 8). The primitive atrium, initially located caudally, moves cephalic and leftward of its original position while the future ventricular and outflow portions move caudally, ventrally, and rightward. Failure of correct rightward looping of the cephalad portions of the cardiac tube results in L-looping of the ventricles, where the morphologic right ventricle is located on the left side of the embryo and ultimately becomes the systemic cardiac pump. Cardiac looping is completed by day 28 (CS 10).

During CS 9, as the embryo folds, the linear heart tube continues to grow, partly through proliferation of the cardiomyocytes but mostly by adding newly differentiated cardiomyocytes, which are derived from the surrounding mesoderm at the poles. During this early stage of relatively restrained cardiomyocyte proliferation in the linear cardiac tube, cells that will make up the bulk of the heart are added from outside the primary tube along its entire length. The two groups of cardiac precursor cells are called the first and second heart fields (see Fig. 5.1 ). The first heart field gives rise primarily to the left ventricle and part of the atria with the right ventricle and outflow tract being derived from the second heart field. It should be noted that the outflow tract (see Fig. 5.1 ) and venous pole of the heart are both frequently involved in congenital heart defects. Therefore, understanding in detail the contribution and timing of the secondary heart field to cardiac development could shed considerable insight into the mechanisms underlying defects such as tetralogy of Fallot and DiGeorge syndrome. The migration and contributions of the primary and secondary heart field cells to the anatomy of the developing heart have been reviewed in detail, and are briefly summarized in Fig. 5.1 . Anatomically and functionally, the heart wall is normally divided into three layers with contractile function restricted to the myocardium, the middle layer of the heart wall, and flanked on the exterior by the epicardium, and the interior by the endocardium. The epicardium, vascular endothelium, and smooth muscle cells arise from the proepicardial organ (see Fig. 5.1 ), which exists transiently as an extracardiac cluster of cells arising as an outgrowth of the coelomic mesothelium at the ventrocaudal base of the developing heart. This process is considered in more detail in the “Fibroblast” section below.

Although briefly outlined in Fig. 5.1 , a complete description of the lineage tree for myocardial cells, their development, and the formation of the anatomic structures is beyond the scope of this chapter. For example, cardiac neural crest contributions are essential for formation of cardiac structures, such as the endocardial cushions (see Fig. 5.1 ) and the smooth muscle component of the pharyngeal arch arteries, and the reader is referred to several excellent reviews on these topics.

Atrial and Ventricular Septation

With the migration of the first and second heart fields completed, distinct delineation into atria and ventricles begins with the development of thin endocardial cushions at approximately 28 days (CS 10). The primitive cushions become increasingly filled with dense material as development continues. Atrial septation begins at 34 days postovulation (CS 14) with the appearance of a muscular shelf from the roof of the atrial component of the heart tube. This primary atrial septum shelf has a mesenchymal “cap” and grows between the systemic and pulmonary venous openings. The mesenchymal cap of the muscular shelf will eventually fuse with the superior endocardial cushions. The inferior portion of the atrial septum (the atrial spine) develops from mesenchyme approaching inferiorly from the posterior mediastinum, and likewise possesses a cap that will fuse with the primary muscular septum and subsequently with the superior and inferior endocardial cushions. These movements serve to close the primary atrial foramen (ostium primum), the gap between the leading edge of the primary septum and the endocardial cushions. The secondary atrial foramen (ostium secundum) is formed by the breakdown of tissue at the superior margin of the muscular primary septum. By 42 days (CS 18), the septum primum, septum secundum, and foramen ovale are formed.

Ventricular septation is both initiated and completed after the same activities in atrial septation, from 38 days (CS 16) to approximately 50 days (CS 22). The future left and right ventricular chambers (LV and RV, respectively) may be appreciated by 38 to 40 days, with the beginning of the muscular interventricular septum occurring concomitantly with the appearance of the apical portions of the LV and RV. By 42 days (CS 18), the muscular ventricular septum reaches from the floor of the ventricles toward the cardiac crux and the LV outlet portion is closed. At this point, the relatively large interventricular foramen allows communication between the ventricles and indeed is the connecting entrance to the RV. Ventricular septation is completed at approximately 50 days (CS 22) with closure of the inlet septum at the level of the atrioventricular valves.

Formation of Atrioventricular Valves

Following the initiation of endocardial cushion development at approximately 28 days, the atrioventricular canal becomes increasingly demarcated by the endocardial cushions, which are more apparent at 32 days postovulation (CS 13), although cellular content of the cushions is limited. Cushion density is increased by 34 days postovulation (CS 14), coincident with the fusion of the mesenchymal portions of the primary atrial septum with the superior atrioventricular cushion and the atrial spine with the inferior cushion. The atrioventricular junction can be seen at approximately 38 days (CS 16) with separate atrioventricular valves present by 42 days (CS 17 to 18). At this stage, the valve leaflets are still thick but will undergo remodeling to become thinner structures by approximately 56 days (CS 23).

Outflow Tract and Semilunar Valve Development

Outflow track septation and semilunar valve development begins with swelling of the truncal cushions at approximately 36 days postovulation (CS 15). Spiraling of aorticopulmonary septum is seen at 38 days (CS 16), with septation of the truncus arteriosus proceeding from distal to proximal. Development of the semilunar valves begins with endothelial to mesenchymal transformation of the endocardium to form cushions. Distinct but relatively thick semilunar valves are present at approximately 42 days (CS 17 to 18) and will eventually undergo extracellular matrix remodeling to become thin, stratified leaflets—a process that for mammals continues even after birth.

Comparison of Human and Mouse Cardiac Development

Zebrafish and chick models have been very useful in uncovering the lineages, the cell migration, and the anatomic timeline for cardiac development; they have the advantage of easily accessible structures that can be physically and molecularly manipulated. However, for a mammalian, a genetically accessible and experimentally affordable model system, the mouse heart most closely resembles the mature human heart and thus is widely used to investigate mechanisms of normal and perturbed cardiac development. Given the short gestation period of mice, the complex events involved in developing a four-chambered heart are understandably condensed. Thus events occurring over an approximately 4-week period in humans are accomplished in roughly 5 days in mice. The general order of events required to develop from an unlooped heart tube to a septated four chamber heart with four cardiac valves is the same in mouse and human, with atrial septation preceding ventricular septation and outflow tract development. In both species, development of the atrioventricular valves is a relatively long process. Because of the extensive use of the mouse heart in modern cardiovascular experimentation, a general comparison of the major events comprising human and mouse heart development is shown ( Fig. 5.2 ).

Origins of Structural Heart Disease

Many events in cardiac development occur simultaneously, resulting in common constellations of cardiac malformation. Truncus arteriosus occurs due to incomplete septation of the outflow tracts into a separate aorta and pulmonary artery from days 36 to 42 (CS 15 to 18), which in turn alters semilunar valve development and closure of the ventricular septum in the LV outflow region (days 38 to 42, CS 16 to 18). In some congenital heart defects, events early in cardiac developments do not necessarily affect subsequent events. For example, the genesis of “typical” congenitally corrected transposition of the great arteries (also referred to as L-TGA due to the leftward and anterior position of the aorta) is inappropriate looping of the primitive heart tube early in development (days 23 to 28, CS 8 to 10). This event results in a morphologic RV being situated on the left side of the body, and the morphologic LV residing on the right. Atrial situs is not affected, and the great arteries, while not having a normal anterior/posterior relationship, will nevertheless come to reside over the left-sided ventricle in the case of the aorta and over the right-sided ventricle for the pulmonary artery. As a result, the right-sided but morphologic LV will conduct desaturated systemic venous return to the low resistance pulmonary bed, and the left-sided but morphologic RV will conduct highly saturated pulmonary venous blood to the high resistance systemic circulation. While this is “corrected” circulation in the sense that the desaturated and saturated blood is routed into the appropriate vascular bed, when subjected to systemic pressure over the course of decades the morphologic RV is prone to dysfunction. In contrast, D-transposition of the great arteries is due to incomplete spiraling of the pulmonary and aortic trunks, resulting in connection of the aorta to the RV, and the pulmonary artery to the LV. Ventricular septal defects are commonly associated with D-transposition of the great arteries, consistent with the overlap in development of the ventricular outflow tract and ventricular septum. Many of these congenital defects have been elegantly modeled using sophisticated three- and four-dimensional renderings and are available online through mobile applications.

Cardiomyocyte

When we think of the heart and then of the myocardium, the first cell type that comes to mind is the cardiomyocyte even though nonmyocyte cells constitute the majority of cells in terms of number. The cardiomyocytes form the muscular walls of the atria and ventricles, and are derived mainly from the mesodermal cells present in the first and second heart fields. By volume, cardiomyocytes are the predominant cell type in the heart. While it had previously been thought that they were the clear majority of cell type present, better technical methods for marking the distinct cell types have more recently determined that they account for between 25% and 35% of all cardiac cells. These highly specialized cells form the contractile basis of the heart and are filled with sarcomeres, the unitary contractile apparatus, as well as mitochondria, the Ca ²⁺ handling machinery and specialized protein-based macromolecular structures that can propagate rapid, directed, and unitary transmission of electrical signals among the cardiomyocytes, enabling the heart to function as a syncytium. Underlying these specialized functions, the cardiomyocyte is characterized by a well-defined spectrum of cardiac specific muscle proteins, including the cardiac myosins, cardiac actin, cardiac myosin binding protein C, phospholamban, various channel proteins, and others. The cardiac cardiomyocytes are further specialized, with unique cellular protein complements for the atrial and ventricular chambers, and distinct cardiomyocyte subtypes populating the heart's conduction system. Atrial and ventricular cardiomyocytes have different contractile properties and electrophysiologic conduction profiles as well, making them highly specialized functional cells that occupy unique anatomic and functional niches in the heart. They are largely postmitotic and have limited regenerative capabilities. Although human cardiomyocytes are replaced at a low, but significant rate, regenerative capacity is clearly limited and cannot compensate when there is massive cell loss, such as occurs with a myocardial infarction.

Fibroblasts

The fibroblasts form the connective matrix of the myocardium. Cardiomyocyte health and function are inextricably linked to this supportive, extracellular matrix. Classically, the fibroblast has been rather ill-defined as a connective tissue cell derived from the primitive mesoderm. In situ in the myocardium, fibroblasts are visualized as small-sized, flattened cells that lack a basement membrane ( Fig. 5.3 ). They are literally tightly squeezed between, above, and below the cardiomyocytes, and they extend multiple cellular processes that form a dense network consistent with their main function, which in the healthy heart is to generate and maintain the extracellular matrix by producing the fibrillar collagens type I and II. This matrix is largely responsible for the three-dimensional architecture of the heart that is necessary for a normally functioning syncytium as well as fast and regular electrical activation. Considering its importance in both normal and pathogenic processes, it is striking how little we really know about the cardiac fibroblast. However, we are beginning to develop the tools necessary for its characterization in the form of relatively selective or specific markers that will allow lineage tracing and functional identification over time. We now know that prior to scar formation and beneficial or pathogenic remodeling processes, the fibroblast is activated to the myofibroblast, with concomitant transcriptional activation of a protein complement, including the matricellular protein periostin ( Fig. 5.4 ). Although the fibrotic process may be initially adaptive and provides the necessary regional strengthening, which is needed to preserve structural integrity in an injured myocardial wall, the myofibroblast population may continue to proliferate and differentiate, leading to additional scarring and pathogenic remodeling processes (see Figs. 5.3 and 5.4 ). The signaling pathways that underlie these processes and control both the activation and inactivation of myofibroblasts in response to injury are currently subject to intense investigation. While fibroblasts have often been thought of as being electrical “insulators,” the situation appears to be more nuanced. Current can be propagated, and specifically in the myofibroblast the cation-conducting channels are relatively insensitive to changes in voltage or remain open, resulting in a moderately polarized membrane (−20 to −40 mV). Improved optical and electrical recording technologies allowed conduction in cocultures of myofibroblasts and cardiomyocytes, confirming that heterocellular gap junctions developed. The electrophysiology of fibroblasts remains a field very much in flux.

The diverse cellular origins ascribed to the fibroblast population underlies and reflects the difficulty in defining cell markers that are absolutely restricted to fibroblasts. It is now generally accepted that the majority of these cells arise from the proepicardial organ (see Fig. 5.1 ), which subsequently serves as a source for the migratory cells that cover the developing heart and form the embryonic epicardial layer, and that contribute to the vascular endothelium. These epicardial cells support cardiomyocyte proliferation in the developing heart tube and provide the precursors for what will become fibroblasts and vascular smooth muscle cells. Some of these cells undergo an epithelial-to-mesenchymal transition, peel off from the epicardium and populate the atrial and ventricular walls, where, in the latter compartments, they are necessary for the formation of the compact myocardium. These cardiac fibroblasts continue to proliferate, essentially doubling in number during the postnatal period. They form the underlying myocardial scaffold and actively signal to and with the other cellular populations, including the cardiomyocytes, to actively proliferate at the appropriate developmental times or in times of cardiac stress.

Data defining the percentage of fibroblasts in the total cardiac cell population have varied widely with some investigators concluding that as many as 40% to 50% of the cells in the heart may be fibroblasts, although the majority of estimates place the percentages between 25% and 35%. Recent data from the mouse, using carefully defined, multiple markers and sophisticated lineage tracing techniques, have come up with surprisingly low percentages, with the fibroblasts comprising only approximately 20% of the nonmyocyte cells in the mouse heart or 12% to 15% of the total cells. These data have yet to be independently verified, but the investigators analyzed adult human cardiac tissue as well and those data mirrored the murine results.