The heart is the first functional organ to form during the development of an embryo, which, in humans, occurs during the third week post-fertilization. It primarily involves the following sequence of events: the specification of cardiac precursor cells from pluripotent embryonic stem cells and their differentiation into cardiac progenitor cells or cardiomyocytes, the organization of cardiomyocytes into a cardiac tube, followed by septation of the cardiac tube into four chambers as well as the paired arterial trunks. In the past two decades, a plethora of studies using advanced technologies and suitable animal models have provided a detailed account of the various cellular and molecular changes that take place during cardiomyocyte differentiation, the formation of the heart tube, and the development of the four cardiac chambers.
Cardiomyocytes are the chief cell type in the heart and their coordinated contraction as a mass is responsible for the pumping of blood around the developing embryo. Cardiac specification occurs very early on during embryonic development. For instance, in mouse embryos this occurs as early as day 6.5 post fertilization and involves the development of a population of cardiac precursor cells in the lateral posterior epiblast just prior to formation of the primitive streak – a structure which marks the beginning of gastrulation. During gastrulation, as the epiblast cells migrate through the primitive streak, they undergo epithelial-mesenchymal transition and form the mesoderm. Following the induction of mesoderm, the cardiac precursor cells become irreversibly specified into cardiomycytes, and this process of cardiac determination is precisely regulated in time and space by an interplay of various signaling pathways, especially those activated by the Wnt, TGF-beta, FGF, or retinoic acid superfamily of extracellular signaling molecules . A stage-specific role of these signaling pathways have been identified through in vivo and in vitro analyses of embryonic stem cells, as seen in the case of Wnt proteins whose activation is essential for the generation of mesodermal germ layer but needs to be subsequently turned off during the differentiation of cardiac progenitor cells from the mesoderm .
The biochemical signaling pathways involved in cardiomyogenesis are directly or indirectly regulated by a number of mechanical signals throughout cardiac development. A classic example is the complex interplay that takes places between E-cadherin/β-catenin mediated mechanosignaling and Wnt/β-catenin mediated chemical signaling. During development, the nuclear translocation of β-catenin, a transcriptional cofactor in the Wnt signaling pathway, is controlled by the extent of cell-cell contacts formed between cells. Before and during early gastrulation, the epiblast layer assumes a tightly packed cellular morphology due to an increase in E-cadherin mediated mechanical linkages between cells. This leads to a corresponding increase in membrane-bound β-catenin and Wnt signaling. The epiblast responds to Wnt signaling from the underlying endoderm, by release of β-catenin from the membrane, which leads to its accumulation in the cytoplasm. Following this, β-catenin translocates to the nucleus and increases the transcription of Wnt-induced genes, including a Wnt inhibitor which promotes cardiac differentiation . Taking advantage of the close association between these two pathways, a number of force aggregation techniques, such as hanging drop, microwell aggregation, and rotary suspension, have been employed to improve cardiac differentiation by altering cell adhesion dynamics .
Other mechanical factors which are known to influence cardiomyocyte differentiation include fluid shear stress, substrate rigidity, and cyclic stretch. Fluid shear stress, which is the frictional force exerted upon cells when fluid flows over them, is experienced very early on during development. Mouse embryonic stem cells (ESCs) subjected to long durations of fluid shear stress exhibit an increase in cardiac tissue specific markers, such as alpha-sarcomeric actin, smooth muscle actin, platelet-endothelial cell adhesion molecule-1, VEGF receptor 2, and smooth muscle protein 22-alpha . A similar induction in the expression of cardiac markers, such as GATA-4 and alpha-MHC, was observed when embryonic stem cells were subjected to cyclic stretching (repetitive stretching and relaxation patterns that accompany cardiomyocyte contractility) . As well as these biochemical changes, the typically elongated structure of cardiomyocytes is believed to be due to the effects of cyclic stretching .
The effects of substrate rigidity on the proliferation and differentiation potential of cells has been clearly established from a number of studies. The findings related to cardiomyocyte differentiation, however, have been contradictory; while one study noted enhanced cardiomyocyte differentiation from stem cells grown on a stiff matrix , another study pointed out that cardiac specification was driven by soft hydrogel substrates . Working along similar lines, another research group that cultured embryonic stem cells on polyacrylamide hydrogel substrates demonstrated that substrate stiffness only influences the early specification of cardiac progenitor cells from the mesoderm and has no effect on the later stages, when the progenitor cells become irreversibly differentiated into cardiomyocytes. Moreover, their findings linked matrices of intermediate stiffness with maximal cardiomyocyte differentiation . As well as influencing cardiomyocyte differentiation, it has also been shown that substrate stiffness can affect their functional properties. Cardiomyocytes cultured on hydrogels with a stiffness similar to that found in the body were produced action potentials with the longest duration compared to softer or harder substrates .
The heart is derived from the splanchnic lateral mesoderm and initially forms as two crescent-shaped endocardial plates inside the evolving pericardial cavity. As the embryo undergoes lateral and cranial folding, the two plates come closer to each other and eventually fuse at the midline, forming a primordial cardiac tube. The linear cardiac tube is composed of an inner endocardial layer and an outer myocardial sheath, which are separated by an acellular, gelatinous matrix called the cardiac jelly. At this stage, the tube can undergo asynchronous, peristaltic contractions and the cardiac progenitor cells will evolve into primitive heart chambers, including the atria, atrioventricular canal and the left ventricle..
The various events during cardiac development are driven by mechanical signals sensed through cellular communication with its immediate environment, which include both the extracellular matrix as well as intercellular cell-cell contacts. These signals can induce the generation of intracellular forces through large-scale cytoskeletal reorganization, which may again influence the mechanical properties of the extracellular environment. For instance, during the process of endocardial tube formation, the movement of the bilateral heart fields towards the embryonic midline involves the migration of lateral mesodermal cells over the underlying endoderm. The migration of the mesoderm is dependent on a morphogenetic event called endodermal shortening, which occurs around the anterior intestinal portal. The large-scale tissue deformations caused by the shortening of endodermal cells generates tension that pulls the mesoderm cells towards the embryonic midline. It is now known that endodermal shortening is mediated by actomyosin-dependent cellular contractility, as these morphogenetic events are disrupted in the presence of blebbistatin, a myosin-II inhibitor .
Another event in cardiac development that requires changes to cell morphology is the ballooning of the heart chambers. This process involves the expansion and bulging of the walls of the heart tube in such a way that the chambers develop into a bean-shaped structure, with the outer walls taking on a convex curvature and the inner walls becoming concave. A study using high resolution imaging to study cell shape changes in Zebrafish embryo hearts has revealed that the cells of the inner wall maintain a cuboidal shape while the cells of the outer wall flatten and elongate, giving rise to the characteristic bean-shape of the heart chambers. Interestingly, distinct cellular morphologies in the inner and outer wall regions was found to arise as a result of net effect of wall shear forces from circulating blood and contractile forces from intracellular cytoskeletal dynamics .
A biomechanical basis has also been posited for the formation of the heart tube based upon the fusion of two primordial epithelial tubes. According to this theory, actin filaments near the apical surfaces of the cardiac progenitor cells contract, so that the cells become wedge-shaped. As the apical surfaces of neighboring cells curve inwards, it leads to the cylindrical bending of a sheet of epithelial cells. These tissue-level deformations are believed to result in the formation of the cardiac tube . In support of this hypothesis, confocal laser scanning microscopy studies have confirmed the arrangement of actin filaments near the apices of early cardiac epithelial cells . Also, during the process of looping, the tubular heart undergoes various mechanical alterations such as bending and rotational movements before attaining a mature S-shape. From a number of studies, it is now posited that while actin polymerization-mediated shape changes in cardiac epithelial cells drives the bending of the heart tube, the rotational movements are driven by a combination of forces exerted on the heart tube by its encapsulating membrane called the splanchnopleure as well as from contraction of the intracellular cytoskeleton .
Although cardiac contractions begin very early in the evolving heart tube and blood flow is established soon after looping, the circulating blood does not yet serve the function of transporting gases, nutrients, and metabolic wastes within the developing embryo, which at this stage is still carried out by passive diffusion. However, the hemodynamic forces generated from the circulating blood was found to be essential for inducing proper cardiac development and active remodeling of the cardiac vessels, including the valves and the great blood vessels. This is evident from presentations of cardiac abnormalities in Zebrafish embryos, such as diminished looping and impaired valve formation, when either the inflow or the outflow tracts were occluded to reduce wall shear stress . Given the significant role that biomechanical factors such as hemodynamic forces play in controlling cardiac morphogenesis, it is not surprising that a number of congenital cardiac diseases, such as the bicuspid aortic valve disease (BAV) and hypoplastic left heart syndrome (HLHS) are linked to abnormal flow patterns of circulating blood .
It is essential to understand the mechanistic details of cardiomyocyte differentiation and heart development in order to gain insights into the pathology of heart diseases, which account for almost 30% of global death . One such major cardiovascular disease, myocardial infarction, involves a significant loss of cardiomyocytes due to oxygen and nutrient deficiency. However, development of effective therapies for cardiac tissue repair in such diseases remain challenging owing to the non-proliferative nature and extremely low regenerative potential of adult cardiomyocytes. This has resulted in massive research efforts geared towards generating cardiomyocytes from human embryonic stem cells as well as induced pluripotent stem cells under in vitro conditions, which could be used to replenish cardiomyocyte number and volume post cardiac insult .