Vulnerability of the Developing Heart to Oxygen Deprivation as a Cause of Congenital Heart Defects

Abstract

BackgroundThe heart develops under reduced and varying oxygen concentrations, yet there is little understanding of oxygen metabolism in the normal and mal‐development of the heart. Here we used a novel reagent, the ODD‐Luc hypoxia reporter mouse (oxygen degradation domain, ODD) of Hif‐1α fused to Luciferase (Luc), to assay the activity of the oxygen sensor, prolyl hydroxylase, and oxygen reserve, in the developing heart. We tested the role of hypoxia‐dependent responses in heart development by targeted inactivation of Hif‐1α.Methods and ResultsODD‐Luciferase activity was 14‐fold higher in mouse embryonic day 10.5 (E10.5) versus adult heart and liver tissue lysates. ODD‐Luc activity decreased in 2 stages, the first corresponding with the formation of a functional cardiovascular system for oxygen delivery at E15.5, and the second after birth consistent with complete oxygenation of the blood and tissues. Reduction of maternal inspired oxygen to 8% for 4 hours caused minimal induction of luciferase activity in the maternal tissues but robust induction in the embryonic tissues in proportion to the basal activity, indicating a lack of oxygen reserve, and corresponding induction of a hypoxia‐dependent gene program. Bioluminescent imaging of intact embryos demonstrated highest activity in the outflow portion of the E13.5 heart. Hif‐1α inactivation or prolonged hypoxia caused outflow and septation defects only when targeted to this specific developmental window.ConclusionsLow oxygen concentrations and lack of oxygen reserve during a critical phase of heart organogenesis may provide a basis for vulnerability to the development of common septation and conotruncal heart defects.

Disruption of spatiotemporal hypoxic signaling causes congenital heart disease in mice

“…It is generally believed that both environmental and genetic factors based on variations in many different genes contribute to CHD. Environmental or nongenetic risk factors include diabetes mellitus, obesity, and hypoxic responses, but the molecular events driving CHD have remained enigmatic (3)(4)(5)(6). Small observational groups and potential confounding effects have complicated analysis of the contribution of specific environmental effects in human beings.…”

“…HIF1α has profound effects on different molecules regulating the behavior of stem and/or progenitor cells. Genetic inactivation of Hif1a during early but not late developmental stages causes CHDs, suggesting a critical role of hypoxia responses for normal heart development, although the precise mechanisms of the action of HIF1α and the definition of its targets in this context have not been worked out (4).…”

Inactivation of maternal Hif-1α at mid-pregnancy causes placental defects and deficits in oxygen delivery to the fetal organs under hypoxic stress

“…For maternal conditional inactivation of Hif-1α (MATcKO), timed matings were performed with Hif-1α f/f ,β-actinCre + females and ODD-Luc males ( Hif-1α +/+ ) with the presence of a vaginal plug in the morning counted as E0.5. Pregnant dams were treated with TM (3mg/40g bw; Sigma # T5648) in sunflower oil on E8.5 and 9.5 to induce the activity of Cre recombinase as previously described (Hayashi and McMahon, 2002; Kenchegowda et al, 2014). Mice were genotyped by standard PCR methods using primers as described on the Jackson Laboratory website.…”

“…Tissues were homogenized in 1X Cell Culture Lysis Reagent (Promega) and clarified by centrifugation. 10–20µl of the lysate was transferred to a 96 well plate and firefly luciferase activity measured as per manufacturer’s instructions (Promega) using a microplate reader (Flex Station 3, Molecular Devices) as previously described (Kenchegowda et al, 2014). Protein concentrations in the lysate were measured using the Bio-Rad Protein Assay Dye Reagent with BSA as the protein standard.…”

“…To circumvent these limitations, conditional approaches have been used to inactivate Hif genes in specific cell types or at specific stages of development (reviewed in (Bishop and Ratcliffe, 2015)). In a prior study, we showed that Tamoxifen-inducible Cre -mediated cKO of Hif-1α at E9.5 caused defects in the formation of the structures of the cardiac outflow tract, modeling human congenital heart defects (CHD) (Kenchegowda et al, 2014). However, the Tamoxifen-inducible Cre was under the control of the pan-active β-actin promoter, raising the possibility that inactivation of Hif in other organs, in particular the placenta, could contribute to the formation of the structural heart defects.…”