| The outflow tract myocardium and other regions corresponding to the location of the major coronary vessels of the developing chicken heart, display a high level of hypoxia as assessed by the hypoxia indicator EF5. The EF5 positive tissues are also specifically positive for nuclear-localized hypoxia inducible factor-1 alpha (HIF-1alpha), the oxygen-sensitive component of the hypoxia inducible factor-1 (HIF-1) heterodimer. In this study we altered ambient oxygen levels (hypoxia 15%; hyperoxia 75-40%) during developmental stages (ED 4.5-9, HH 25-35) critical to avian coronary vessel development in order to alter tissue hypoxia, HIF-1alpha protein expression and some of its downstream targets. We also altered gene expression in the embryonic outflow tract myocardium by injecting an adenovirus containing a constitutively active form of HIF-1alpha (AdCA5). Under all experimental conditions, HIF-1alpha reached significant levels. Downstream targets of HIF-1alpha, such as VEGF, PDGF-B and VEGF receptor 2 (VEGFR2), were significant after exposure to the experimental conditions as well. We assayed for coronary anomalies and used 3D reconstructions to specifically identify altered morphology with the coronary vasculature. When incubated for 4.5 days under abnormal oxygen levels or injected with the AdCA5, coronary arteries displayed 13 deviations from their normal proximal connections to the aorta. These deviations were similar to known clinical anomalies of coronary arteries. These findings indicate that developing coronary vessels may be subject to a level of regulation that is dependent on differential oxygen levels within cardiac tissues and subsequent HIF-1 regulation of gene expression. |
