We proposed a model in which myocardial hypoxia triggers the apoptosis-dependent remodeling of the avian outflow tract (OFT) in the transition of the embryo to a dual circulation. In this study, we examined hypoxia-dependent signaling in cardiomyocyte apoptosis and outflow tract remodeling. The hypoxia-inducible transcription factor HIF-1alpha was specifically present in the nuclei of OFT cardiomyocytes from stages 25-32, the period of hypoxia-dependent OFT remodeling. HIF-1alpha expression was sensitive to changes in ambient oxygen concentrations, while its dimerization partner HIF-1beta was constitutively expressed. There was not a simple relationship between HIF-1alpha expression and apoptosis. Apoptotic cardiomyocytes were detected in HIF-1alpha-positive and -negative regions, and a hypoxic stimulus sufficient to induce nuclear accumulation of HIF-1alpha did not induce cardiomyocyte apoptosis. The hypoxia-dependent expression of the vascular endothelial growth factor receptor (VEGFR2) in the distal OFT myocardium may be protective as cardiomyocyte apoptosis in the early stages (25-30) of OFT remodeling was absent from this region. Furthermore, recombinant adenoviral-mediated expression of dominant negative Akt, an inhibitor of tyrosine kinase receptor signaling, augmented cardiomyocyte apoptosis in the OFT and constitutively active Akt suppressed it. Adenovirus-mediated forced expression of VEGF165 induced conotruncal malformation such as double outlet right ventricle (DORV) and ventricular septal defect (VSD), similar to defects observed when apoptosis-dependent remodeling of the OFT was specifically targeted. We conclude that normal developmental remodeling of the embryonic avian cardiac OFT involves hypoxia/HIF-1-dependent signaling and cardiomyocyte apoptosis. Autocrine signaling through VEGF/VEGFR2 and Akt provides survival signals for the hypoxic OFT cardiomyocytes, and regulated VEGF signaling is required for the normal development of the OFT.

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.

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