Cardiac development is an intrinsically multiscale problem. An exquisite orchestration of preprogrammed genetic events prompts cells to proliferate, differentiate, migrate, apoptose, and secrete ECM proteins at the right time and in the right place, giving rise to morphogenetic events that end with heart formation. Genetic programs, however, are modulated by mechanical stimuli. This modulation allows the embryo to survive harsh in utero conditions but can also lead to cardiac malformations. To fully understand the origins of congenital heart disease (CHD), therefore, it is necessary to study the heart and its mechanics as it develops.
The heart is the first functional organ. Early during embryonic development (about 3 weeks of gestation in humans) a primitive, linear tubular heart is formed. Soon after formation, the tubular heart starts beating and pumping blood by a peristaltic-like mechanism (similar to a peristaltic pump). The heart then bends and twists, forming a looping heart tube. At these initial stages of heart formation, cardiac walls consist of a thin layer of contractile myocardial cells (the myocardium), a monolayer of ECs in contact with blood flow (the endocardium), and an ECM layer in between the myocardium and the endocardium (the cardiac jelly). Further, different cardiac structures start taking shape in the tubular heart.
From inlet to outlet, the tubular heart consists of a primitive atrium, an atrioventricular (AV) canal, a ventricle, and an OFT. During tubular heart stages, endocardial cushions, which are precursors of valves and a small portion of the interventricular septum, develop in the AV canal and heart OFT. The cushions are basically non-uniform thickenings of cardiac jelly in the heart wall (typically two opposing cushions) and initially function as primitive valves by closing the lumen upon myocardial contraction. An endothelial-mesenchymal transition (EMT) then takes place, first in the AV cushions and then in the OFT cushions. During EMT, ECs detach from the endocardial layer, acquire a migration phenotype, and start migrating into the cushions. Migrating cells populate the cushions and change the cardiac jelly ECM composition by secreting different proteins as they move and proliferate. Endocardial cushion remodeling, which involves increases in cell density and changes in ECM composition, precedes valve formation and cardiac septation. Thus, appropriate EMT progression is required for proper heart formation. By the end of the first trimester (about 50 days of gestation in humans) the heart is fully formed, but it continues to grow and mature in preparation for birth. This last maturation step involves increase in the number of cells, in particular myocardial cells, and maturation of the myocardial contractile system by careful organization of myofibrils and mitochondria to achieve maximum cardiac efficiency.
Congenital cardiac defects occur in about 1% of newborn babies, and they are the leading cause of non-infectious deaths in babies and children in the developed world. CHD is also the most common type of congenital malformation in babies. This is in a way not entirely surprising, given the complex processes that give rise to heart formation.
A number of genes and chromosome mutations have been associated with cardiac defects. For example, a mutation in JAGGED-1 has been associated with tetralogy of Fallot (TOF) , as well as deletion of chromosome 22qll.2 ; a mutation in NKX2.5 was associated with atrial septal defect  ; mutations in ZIC3 and TBX20 were associated with double-outlet right ventricle (DORV) [22, 23]; and a mutation in MYH7 was associated with ventricular septal defects (VSDs) . Mutations of several other genes that play important roles in cardiac development have also been linked to CHD, including mechanotransduction genes [21-23, 25, 26].
In addition to gene and chromosomal mutations, exposure to teratogens during gestation has also been associated with cardiac malformations. For example, prenatal exposure to ethanol and retinoic acid has been associated with TOF and DORV , while exposure to theophylline, a drug used to treat respiratory diseases, is associated with DORV, transposition of the great arteries and hypoplastic left ventricle . Meanwhile, maternal nutrition can affect heart formation , as can maternal diabetes [29, 30], with maternal diabetes predominantly leading to DORV and truncus arteriosus .
The preceding discussion shows that it is relatively easy to alter the course of normal heart development, leading to cardiac malformations. Gene mutations and teratogens can certainly alter cardiac development. Perhaps less evident, blood flow can also alter cardiac development. Blood flow is an integral part of heart development, constantly providing mechanical feedback to ensure proper cardiac formation and function. In this way, we can think of the blood flow feedback as a mechanism regulating cardiac development. When mechanical feedback is anomalous, malformations can develop.
Effect of Blood Flow on Cardiac Formation
Cardiac development is the result of genetic programs modulated by in utero environmental factors [32-35], including biomechanical factors. Abnormal cardiac biomechanics, for example, due to abnormal blood flow conditions caused by placental anomalies or the mother's obesity, result in the formation of heart defects, and thus CHD [36, 37]. The vast majority of CHD cases do not present familial history, and genetic tests are normal . Many researchers now believe that most CHD cases are the result of complex etiologies that include environmental in utero exposure (e.g., smoking, diabetes, and placenta anomalies). Abnormal blood flow conditions during critical embryonic developmental stages are now accepted to be the likely cause of many CHD cases.
The heart pumps blood from its initial tubular stages. Cardiac morphological processes, such as cardiac looping, endocardial cushion formation, EMT, valve formation, and cardiac septation, thus, occur while cardiac tissues are interacting with blood flow dynamics. Blood flow influences any of these morphological processes.
The importance of blood flow in heart development is supported by mutant mice and zebrafish models [23, 26]. Mutations affecting cardiac contractile proteins, which impact blood flow, present defects of endocardial cushions. For example, mouse models with mutations of cardiac troponin T and Na2 - Ca2 exchanger (Ncxl) that prevent the heart from beating show endocardial cushion defects and ventricular underdevelopment. Further, mutations affecting known flow mechanotransduction genes (ET-1, NOS3, KLF2, etc.) also show defects associated with abnormal endocardial cushion formation; and mutations in transcription factors (NKX2.5, GATA4, TBX1, TBX5, etc.) lead to cardiac malformations, with similar defects found after altering hemodynamic conditions [6,19, 23, 26, 39-42]. The details of the mechanisms involved in mechanical feedback during heart formation are only starting to emerge. Different animal models are currently being used to better understand mechanotransduction mechanisms during heart development.
Animal Models of Cardiac Development
Several animal models can be used to study cardiac development.
Each model has its own advantages and disadvantages, and therefore different models are used depending on the specific aspect being investigated. Among mammal models, perhaps the most widely used animal model of cardiac development is the mouse. Because genetic manipulations on mouse models are common practice, these models have been extensively used for studying the effects of different genes on cardiac formation, and even for tracing cell lineages [43, 44]. The main disadvantage of mouse models is that it is very difficult to image cardiac development and cardiac function in vivo as non-invasive imaging techniques do not have the resolution and penetration needed. In addition, the placenta of the mouse is very different from that of humans, making the mouse a non-ideal model to study placenta-cardiovascular interactions in the embryos. Guinea pig models, on the other hand, are excellent models for studying the influence of the placenta on embryonic development and heart formation [45,46]. This is because the guinea pig placenta is very similar to human placentas. As in the case of mouse models, cardiac development in guinea pig embryos is difficult to follow in vivo. Moreover, because guinea pig models are not widely used, and genetic modifications as well as specific antibodies are not generally available, guinea pig models are barely used in cardiac developmental studies. In both guinea pig and mouse models, in addition, measurement of blood flow dynamics during embryonic development is not possible without disturbing the embryo and its environment; and manipulations to alter blood flow conditions, such as surgical interventions, are also challenging. Recently, embryonic cardiac imaging on mouse embryos was measured using optical coherence tomography (OCT), with excellent resolution . OCT imaging required externalization of embryos from the mother, restricting embryo viability over time. Nevertheless, these new developments are promising to study early deviations of blood flow conditions in genetically modified mice.
Other mammalian models used to study cardiac development are sheep and nonhuman primates. Sheep are an excellent model to study late gestation, as the fetus is large enough to instrument its cardiovascular system and allow hemodynamic manipulations [48- 51]. Moreover, the effect of drugs and hormones can be studied by injection in the fetus or mother . Due to cost issues, nonhuman primates are mainly used in long-term studies, such as studies to determine influences of obesity and mother nutrition on offspring cardiovascular health . Study of early embryonic development in sheep and primates poses the same difficulties as those faced in the study of early embryonic development in mice models: access to the embryos for in vivo imaging and manipulation is challenging. While large mammalian models are promising in the study of cardiovascular development, the expenses associated with them are large, and thus only a few labs pursue studies in those models.
Avian and zebrafish models of development are all-time favorites in the study of early embryonic cardiac development. This is because the embryos are transparent, enabling high-resolution optical imaging (confocal, light sheet, OCT, etc.) and easily accessible for in vivo imaging and manipulation. Further, since genetic processes are highly conserved among vertebrate species, cardiac developmental processes relevant to human development can be elucidated using avian and zebrafish models. Genetic manipulations are available in zebrafish, allowing scanning of diverse mutations [54-57]. Hemodynamic manipulations in zebrafish embryos, other than through drugsand compounds, are generally very difficultto perform, somewhat restricting the range of hemodynamic studies that can be performed in zebrafish. Moreover, unlike humans, the zebrafish heart has two chambers, restricting the range of heart defects that can be studied. Nevertheless, given the ease of in vivo imaging, fast developmental time, and moderate costs of establishing zebrafish colonies, zebrafish embryos are promising models extensively used in cardiac developmental studies.
Avian models (typically chicken) are also extensively used in cardiac developmental studies. This is due to the ease with which avian embryos can be accessed within the eggs without affecting cardiac growth or function, the ease of embryo manipulation, and cost-effectiveness. Since the circulation is readily accessible, topical application of drugs directly onto the heart or by injection into the circulation is common practice . Further, heart development in humans and chicks is very similar. Like the human heart, the fully formed chicken heart has four chambers and valves, enabling recapitulation of human defects. The avian embryo, moreover, allows for relatively easy monitoring of different parts of its cardiovascular system in vivo data collection. Hemodynamic manipulations, through surgical interventions, are relatively easy to perform in avians and common practice, making the avian model ideal for altering flow patterns at will and thus for studies on the influence of blood flow on cardiac development.
Several surgical interventions have been performed in chicken embryos to alter blood flow conditions at early embryonic developmental stages [36, 59-61]. These interventions mimic changes in hemodynamic conditions due to several factors, such as genetic anomalies and teratogen exposure and also placenta dysfunction and vitelline anomalies, both of which affect embryonic blood flow dynamics (see Fig. 5.3 A). Two of the most extensively used interventions are (i) vitelline vein ligation (WL; Fig. 5.3C), in which blood flow through the right or left vitelline vein, which returns blood to the heart, is blocked, reducing blood flow through the heart and decreasing wall shear stress [61, 62]; and (ii) OFT banding (OTB; Fig. 5.3 D), in which a suture is placed and tightened around the heart OFT, increasing blood pressure in the heart and wall shear stress in the OFT [63-65]. OTB results in a range of hemodynamic perturbations that depend on the band tightness or degree of suture constriction of the OFT walls [66, 67]. Band tightness, calculated as the percent difference of the change in maximum OFT diameter before and after banding, can be used as both a measure of OFT constriction and the level of blood flow perturbation.
Figure 5.3 Abnormal hemodynamics. (A) Human embryonic circulation with factors contributing to hemodynamic perturbations. (8) Schematic of a normal chick embryo at day В (HH18), corresponding to about 4 weeks in human development. Sample optical images of chick embryos at HH18, after surgical interventions used to alter blood flow with (C) vitelline vein ligation (VVL) and (D) outflow tract banding (OTB). Scale bar = 1 mm .
Studies that alter normal cardiac blood flow dynamics are elucidating important mechanotransduction processes and mechanisms by which blood flow dynamics influences heart development. Studies aiming at understanding the effects of blood flow dynamics on cardiac development can be generally divided into two groups: (i) studies that focus on early heart changes due to altered blood flow conditions and (ii] studies that focus on the resulting heart malformations later during development, when the heart is fully formed.