Role of myocardial hypoxia in the remodeling of the embryonic avian cardiac outflow tract

The embryonic cardiac outflow myocardium originates from a secondary heart-forming field to connect the developing ventricles with the aortic sac. The outflow tract (OFT) subsequently undergoes complex remodeling in the transition of the embryo to a dual circulation. In avians, elimination of OFT cardiomyocytes by apoptosis (stages 25-32) precedes coronary vasculogenesis and is necessary for the shortening of the OFT and the posterior rotation of the aorta. We hypothesized that regional myocardial hypoxia triggers OFT remodeling. We used immunohistochemical detection of the nitroimidazole EF5, administered by intravascular infusion in ovo, as an indicator of relative tissue oxygen concentrations. EF5 binding was increased in the OFT myocardium relative to other myocardium during these stages (25-32) of OFT remodeling. The intensity of EF5 binding paralleled the prevalence of apoptosis in the OFT myocardium, which are first detected at stage 25, maximal at stage 30, and diminished by stage 32. Evidence of coincident hypoxia-dependent responses included the expression of the vascular endothelial growth factor (VEGF) receptor 2 by the OFT myocardium, the predominant expression of VEGF122 (diffusible) isoform in the OFT, and the recruitment of QH1-positive pro-endothelial cells to the OFT and vasculogenesis. Exposure of embryos to hyperoxia (95% O(2)/5% CO(2)) during this developmental window reduced the prevalence of cardiomyocyte apoptosis and attenuated the shortening and rotation of the OFT, resulting in double-outlet right ventricle morphology, similar to that observed when apoptosis is directly inhibited. These results suggest that regional myocardial hypoxia triggers cardiomyocyte apoptosis and remodeling of the OFT in the transition to a dual circulation, and that VEGF autocrine/paracrine signaling may regulate these processes.     

Developmental changes in cardiac recovery from anoxia-reoxygenation

The developing cardiovascular system is known to operate normally in a hypoxic environment. However, the functional and ultrastructural recovery of embryonic/fetal hearts subjected to anoxia lasting as long as hypoxia/ischemia performed in adult animal models remains to be investigated. Isolated spontaneously beating hearts from Hamburger-Hamilton developmental stages 14 (14HH), 20HH, 24HH, and 27HH chick embryos were subjected in vitro to 30 or 60 min of anoxia followed by 60 min of reoxygenation. Morphological alterations and apoptosis were assessed histologically and by transmission electron microscopy. Anoxia provoked an initial tachycardia followed by bradycardia leading to complete cardiac arrest, except for in the youngest heart, which kept beating. Complete atrioventricular block appeared after 9.4 +/- 1.1, 1.7 +/- 0.2, and 1.6 +/- 0.3 min at stages 20HH, 24HH, and 27HH, respectively. At reoxygenation, sinoatrial activity resumed first in the form of irregular bursts, and one-to-one atrioventricular conduction resumed after 8, 17, and 35 min at stages 20HH, 24HH, and 27HH, respectively. Ventricular shortening recovered within 30 min except at stage 27HH. After 60 min of anoxia, stage 27HH hearts did not retrieve their baseline activity. Whatever the stage and anoxia duration, nuclear and mitochondrial swelling observed at the end of anoxia were reversible with no apoptosis. Thus the embryonic heart is able to fully recover from anoxia/reoxygenation although its anoxic tolerance declines with age. Changes in cellular homeostatic mechanisms rather than in energy metabolism may account for these developmental variations.     

Ablation of the secondary heart field leads to tetralogy of Fallot and pulmonary atresia

Recent studies in chick and mouse embryos have identified a previously unrecognized secondary heart field (SHF), located in the ventral midline splanchnic mesenchyme, which provides additional myocardial cells to the outflow tract as the heart tube lengthens during cardiac looping. In order to further delineate the contribution of this secondary myocardium to outflow development, we labeled the right SHF of Hamburger-Hamilton (HH) stage 14 chick embryos via microinjection of DiI/rhodamine and followed the fluorescently labeled cells over a 96-h time period. These experiments confirmed the movement of the SHF into the outflow and its spiraling migration distally, with the right side of the SHF contributing to the left side of the outflow. In contrast, when the right SHF was labeled at HH18, the fluorescence was limited to the caudal wall of the lengthening aortic sac. We then injected a combination of DiI and neutral red dye, and ablated the SHF in HH14 or 18 chick embryos. Embryos were allowed to develop until day 9, and harvested for assessment of outflow alignment. Of the embryos ablated at HH14, 76% demonstrated cardiac defects including overriding aorta and pulmonary atresia, while none of the sham-operated controls were affected. In addition, the more severely affected embryos demonstrated coronary artery anomalies. The embryos ablated at HH18 also manifested coronary artery anomalies but maintained normal outflow alignment. Therefore, the myocardium added to the outflow by the SHF at earlier stages is required for the elongation and appropriate alignment of the outflow tract. However, at later stages, the SHF contributes to the smooth muscle component of the outflow vessels above the pulmonary and aortic valves which is important for the development of the coronary artery stems. This work suggests a role for the SHF in a subset of congenital heart defects that have overriding aorta and coronary artery anomalies, such as tetralogy of Fallot and double outlet right ventricle.     

Distinct cardiac malformations caused by absence of connexin 43 in the neural crest and in the non-crest neural tube

Connexin 43 (Cx43) is expressed in the embryonic heart, cardiac neural crest (CNC) and neural tube, and germline knockout (KO) of Cx43 results in aberrant cardiac outflow tract (OFT) formation and abnormal coronary deployment. Prior studies suggest a vital role for CNC expression of Cx43 in heart development. Surprisingly, we found that conditional knockout (CKO) of Cx43 in the dorsal neural tube and CNC mediated by Wnt1-Cre failed to recapitulate the Cx43-null OFT phenotype, although coronary vasculature was abnormal in this mutant line. A broader CKO mediated by P3pro (Pax3)-Cre, involving both ventral and dorsal aspects of the thoracic neural tube and CNC, resulted in infundibular bulging and coronary anomalies similar to those seen in germline Cx43-null hearts. P3pro-Cre-mediated loss of Cx43 in the neural tube was characterized by a late phase of cellular delamination from the dorsal and lateral neural tube, a markedly increased abundance of neuroepithelium-derived cells outside of the neural tube and an excess of such cells infiltrating the heart and infundibulum. Thus, expression of Cx43 in the CNC is crucial for normal coronary deployment, but Cx43 is not required in the CNC for normal OFT morphogenesis. Rather, this study suggests a novel function for Cx43 in which Cx43 acts through non-crest neuroepithelial cells to suppress cellular delamination from the neural tube and thereby preserve normal OFT development.     

Expression of cardiac neural crest and heart genes isolated by modified differential display

The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent outflow tract septation are critical events during vertebrate heart development. We have performed four modified differential display screens in the chick embryo to identify genes that may be involved in CNC, OFT, secondary heart field, and heart development. The screens included differential display of RNA isolated from three different axial segments containing premigratory cranial neural crest cells; of RNA from distal outflow tract, proximal outflow tract, and atrioventricular tissue of embryonic chick hearts; and of RNA isolated from left and right cranial tissues, including the early heart fields. These screens have resulted in the identification of the five cDNA clones presented here, which are expressed in the cardiac neural crest, outflow tract and developing heart in patterns that are unique in heart development.     

Elevated transforming growth factor beta2 enhances apoptosis and contributes to abnormal outflow tract and aortic sac development in retinoic X receptor alpha knockout embryos

Septation of the single tubular embryonic outflow tract into two outlet segments in the heart requires the precise integration of proliferation, differentiation and apoptosis during remodeling. Lack of proper coordination between these processes would result in a variety of congenital cardiac defects such as those seen in the retinoid X receptor alpha knockout (Rxra(-/-)) mouse. Rxra(-/-) embryos exhibit lethality between embryonic day (E) 13.5 and 15.5 and harbor a variety of conotruncal and aortic sac defects making it an excellent system to investigate the molecular and morphogenic causes of these cardiac malformations. At E12.5, before the embryonic lethality, we found no qualitative difference between wild type and Rxra(-/-) proliferation (BrdU incorporation) in outflow tract cushion tissue but a significant increase in apoptosis as assessed by both TUNEL labeling in paraffin sections and caspase activity in trypsin-dispersed hearts. Additionally, E12.5 embryos demonstrated elevated levels of transforming growth factor beta2 (TGFbeta2) protein in multiple cell lineages in the heart. Using a whole-mouse-embryo culture system, wild-type E11.5 embryos treated with TGFbeta2 protein for 24 hours displayed enhanced apoptosis in both the sinistroventralconal cushion and dextrodorsalconal cushion in a manner analogous to that observed in the Rxra(-/-). TGFbeta2 protein treatment also led to malformations in both the outflow tract and aortic sac. Importantly, Rxra(-/-) embryos that were heterozygous for a null mutation in the Tgfb2 allele exhibited a partial restoration of the elevated apoptosis and of the malformations. This was evident at both E12.5 and E13.5. The data suggests that elevated levels of TGFbeta2 can (1) contribute to abnormal outflow tract morphogenesis by enhancing apoptosis in the endocardial cushions and (2) promote aortic sac malformations by interfering with the normal development of the aorticopulmonary septum.     

4D subject-specific inverse modeling of the chick embryonic heart outflow tract hemodynamics

Blood flow plays a critical role in regulating embryonic cardiac growth and development, with altered flow leading to congenital heart disease. Progress in the field, however, is hindered by a lack of quantification of hemodynamic conditions in the developing heart. In this study, we present a methodology to quantify blood flow dynamics in the embryonic heart using subject-specific computational fluid dynamics (CFD) models. While the methodology is general, we focused on a model of the chick embryonic heart outflow tract (OFT), which distally connects the heart to the arterial system, and is the region of origin of many congenital cardiac defects. Using structural and Doppler velocity data collected from optical coherence tomography, we generated 4D (\(\hbox {3D}\,+\,\hbox {time}\)) embryo-specific CFD models of the heart OFT. To replicate the blood flow dynamics over time during the cardiac cycle, we developed an iterative inverse-method optimization algorithm, which determines the CFD model boundary conditions such that differences between computed velocities and measured velocities at one point within the OFT lumen are minimized. Results from our developed CFD model agree with previously measured hemodynamics in the OFT. Further, computed velocities and measured velocities differ by \(<\)15 % at locations that were not used in the optimization, validating the model. The presented methodology can be used in quantifications of embryonic cardiac hemodynamics under normal and altered blood flow conditions, enabling an in-depth quantitative study of how blood flow influences cardiac development.     

An Nkx2-5/Bmp2/Smad1 negative feedback loop controls heart progenitor specification and proliferation

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.     

Misexpression of noggin leads to septal defects in the outflow tract of the chick heart.

BMP-2 and BMP-4 are known to be involved in the early events which specify the cardiac lineage. Their later patterns of expression in the developing mouse and chick heart, in the myocardium overlying the atrioventricular canal (AV) and outflow tract (OFT) cushions, also suggest that they may play a role in valvoseptal development. In this study, we have used a recombinant retrovirus expressing noggin to inhibit the function of BMP-2/4 in the developing chick heart. This procedure resulted in abnormal development of the OFT and the ventricular septum. A spectrum of abnormalities was seen ranging from common arterial trunk to double outlet right ventricle. In hearts infected with noggin virus, where the neural crest cells have been labelled, the results show that BMP-2/4 function is required for the migration of neural crest cells into the developing OFT to form the aortopulmonary septum. Prior to septation, misexpression of noggin also leads to a decrease in the number of proliferating mesenchymal cells within the proximal cushions of the outflow tract. These results suggest that BMP-2/4 function may mediate several key events during cardiac development.     

Myocardialization of the cardiac outflow tract.

During development, the single-circuited cardiac tube transforms into a double-circuited four-chambered heart by a complex process of remodeling, differential growth, and septation. In this process the endocardial cushion tissues of the atrioventricular junction and outflow tract (OFT) play a crucial role as they contribute to the mesenchymal components of the developing septa and valves in the developing heart. After fusion, the endocardial ridges in the proximal portion of the OFT initially form a mesenchymal outlet septum. In the adult heart, however, this outlet septum is basically a muscular structure. Hence, the mesenchyme of the proximal outlet septum has to be replaced by cardiomyocytes. We have dubbed this process "myocardialization." Our immunohistochemical analysis of staged chicken hearts demonstrates that myocardialization takes place by ingrowth of existing myocardium into the mesenchymal outlet septum. Compared to other events in cardiac septation, it is a relatively late process, being initialized around stage H/H28 and being basically completed around stage H/H38. To unravel the molecular mechanisms that are responsible for the induction and regulation of myocardialization, an in vitro culture system in which myocardialization could be mimicked and manipulated was developed. Using this in vitro myocardialization assay it was observed that under the standard culture conditions (i) whole OFT explants from stage H/H20 and younger did not spontaneously myocardialize the collagen matrix, (ii) explants from stage H/H21 and older spontaneously formed extensive myocardial networks, (iii) the myocardium of the OFT could be induced to myocardialize and was therefore "myocardialization-competent" at all stages tested (H/H16-30), (iv) myocardialization was induced by factors produced by, most likely, the nonmyocardial component of the outflow tract, (v) at none of the embryonic stages analyzed was ventricular myocardium myocardialization-competent, and finally, (vi) ventricular myocardium did not produce factors capable of supporting myocardialization.     

Biomechanics of the Chick Embryonic Heart Outflow Tract at HH18 Using 4D Optical Coherence Tomography Imaging and Computational Modeling

During developmental stages, biomechanical stimuli on cardiac cells modulate genetic programs, and deviations from normal stimuli can lead to cardiac defects. Therefore, it is important to characterize normal cardiac biomechanical stimuli during early developmental stages. Using the chicken embryo model of cardiac development, we focused on characterizing biomechanical stimuli on the Hamburger-Hamilton (HH) 18 chick cardiac outflow tract (OFT), the distal portion of the heart from which a large portion of defects observed in humans originate. To characterize biomechanical stimuli in the OFT, we used a combination of in vivo optical coherence tomography (OCT) imaging, physiological measurements and computational fluid dynamics (CFD) modeling. We found that, at HH18, the proximal portion of the OFT wall undergoes larger circumferential strains than its distal portion, while the distal portion of the OFT wall undergoes larger wall stresses. Maximal wall shear stresses were generally found on the surface of endocardial cushions, which are protrusions of extracellular matrix onto the OFT lumen that later during development give rise to cardiac septa and valves. The non-uniform spatial and temporal distributions of stresses and strains in the OFT walls provide biomechanical cues to cardiac cells that likely aid in the extensive differential growth and remodeling patterns observed during normal development.