Mechanism Sharing Between Genetic and Gestational Hypoxia-Induced Cardiac Anomalies

Abstract

Background: Cardiac development is a dynamic process both temporally and spatially. These complex processes are often disturbed and lead to congenital cardiac anomalies that affect approximately 1% of live births. Disease-causing variants in several genetic loci lead to cardiac anomalies, with variants in transcription factor NKX2-5 gene being one of the largest variants known. Gestational hypoxia, such as seen in high-altitude pregnancy, has been known to affect cardiac development, yet the incidence and underlying mechanisms are largely unknown.Methods and Results: Normal wild-type female mice mated with heterozygous Nkx2-5 mutant males were housed under moderate hypoxia (14% O2) or normoxia (20.9% O2) conditions from 10.5 days of gestation. Wild-type mice exposed to hypoxia demonstrate excessive trabeculation, ventricular septal defects, irregular morphology of interventricular septum as well as atrial septal abnormalities, which overlap with those seen in heterozygous Nkx2-5 mutant mice. Genome-wide transcriptome done by RNA-seq of a 2-day hypoxic exposure on wild-type embryos revealed abnormal transcriptomes, in which approximately 60% share those from Nkx2-5 mutants without hypoxia. Gestational hypoxia reduced the expression of Nkx2-5 proteins in more than one-half along with a reduction in phosphorylation, suggesting that abnormal Nkx2-5 function is a common mechanism shared between genetic and gestational hypoxia-induced cardiac anomalies, at least at a specific developing stage.Conclusion: The results of our study provide insights into a common molecular mechanism underlying non-genetic and genetic cardiac anomalies.

Defective coronary vessel organization and reduction of VEGF‐A in mouse embryonic hearts with gestational mild hypoxia

“…In this study, we analyzed coronary vessel organization in embryonic hearts exposed to continuous mild gestational hypoxia (O 2 , 14%) compared to normoxic hearts and found defective coronary vessel organization (Figure ) and a reduction in VEGF‐A mRNA and proteins (Figure 1D‐G). The E15.5 hearts with hypoxia showed a tendency for thinner compact layer, and an increase of trabecular area relative to the compact area (Figure 1B‐C), which is consistent with our previous study showing that newborn mice with gestational hypoxia develop cardiac anomalies, such as ventricular septal defects, excessive ventricular trabeculation, and irregular interventricular septum . These data suggest the link between hypoxia‐induced defective coronary vessel organization and cardiac anomalies.…”

“…A previous study showed that Hif1α was upregulated in E12.5 hearts under severe hypoxia (8% for 6 hours) . Under 14% chronic hypoxia starting from E10.5, however Hif1α mRNA and proteins were not upregulated in embryonic hearts either at 2 days (E12.5) or 8 days (E18.5) of exposure to hypoxia . VEGF‐A expression was most downregulated at E12.5 compared to E15.5, and was not significantly different between hypoxic and normoxic groups at E18.5 (Figure 1D).…”

“…We recently showed that continuous gestational mild hypoxia (14% O 2 ) applied from E10.5 to E18.5 resulted in thin myocardium and cardiac anomalies in newborn 129/Sv mice (Figure 1A). The paraffin‐sectioned E15.5 hearts coimmunostained with an endocardial marker (endomucin—green) and a myocardial marker (troponin T—red) were used for the measurement of compact layer width, as well as area relative to the trabecular and total area.…”

“…In a previous study, we also examined mRNA expression 2 days after hypoxia at E12.5 by RNAseq (raw data are available NCBI GEO GSE114532), in which VEGF‐A mRNA was decreased by 36.5% in hypoxic hearts relative to normoxic controls ( n = 3 or 4 per group). Taqman q‐PCR confirmed the reduction of VEGF‐A mRNA in hypoxic hearts by 65% at E12.5 and by 25% at E15.5, but there was no difference between two groups at E18.5 (Figure 1D).…”

“…The animal models and application of hypoxia was reported in our previous study . In short, wild‐type 129/Sv female mice were placed in a hypoxic chamber (COY Lab Products, Grass Lake, Michigan) that was connected to nitrogen and oxygen gas.…”