Congenital heart disease (CHD) affects almost one percent of all live births. Despite diagnostic and surgical reparative advances, the causes and mechanisms of CHD are still primarily unknown. The extracellular matrix plays a large role in cell communication, function, and differentiation, and therefore likely plays a role in disease development and pathophysiology. Cell adhesion and gap junction proteins, such as integrins and connexins, are also essential to cellular communication and behavior, and could interact directly (integrins) or indirectly (connexins) with the extracellular matrix. In this work, we explore disparities in the expression and spatial patterning of extracellular matrix, adhesion, and gap junction proteins between wild type and Nkx2-5 +/R52G mutant mice. Decellularization and proteomic analysis, Western blotting, histology, immunostaining, and mechanical assessment of embryonic and neonatal wild type and Nkx2-5 mutant mouse hearts were performed. An increased abundance of collagen IV, fibronectin, and integrin β-1 was found in Nkx2-5 mutant neonatal mouse hearts, as well as increased expression of connexin 43 in embryonic mutant hearts. Furthermore, a ventricular noncompaction phenotype was observed in both embryonic and neonatal mutant hearts, as well as spatial disorganization of ECM proteins collagen IV and laminin in mutant hearts. Characterizing such properties in a mutant mouse model provides valuable information that can be applied to better understanding the mechanisms of congenital heart disease.

Cardiac development is a dynamic process, temporally and spatially. When disturbed, it leads to congenital cardiac anomalies that affect approximately 1% of live births. Genetic variants in several loci lead to anomalies, with the transcription factor NKX2-5 being one of the largest. However, there are also non-genetic factors that influence cardiac malformations. We examined the hypothesis that hyperoxia may be beneficial and can rescue genetic cardiac anomalies induced by an Nkx2-5 mutation. Intermittent mild hyperoxia (40% PO2) was applied for 10 h per day to normal wild-type female mice mated with heterozygous Nkx2-5 mutant males from gestational day 8.5 to birth. Hyperoxia therapy reduced excessive trabeculation in Nkx2-5 mutant mice compared to normoxic conditions (ratio of trabecular layer relative to compact layer area, normoxia 1.84 ± 0.07 vs. hyperoxia 1.51 ± 0.04) and frequency of muscular ventricular septal defects per heart (1.53 ± 0.32 vs. 0.68 ± 0.15); however, the incidence of membranous ventricular septal defects in Nkx2-5 mutant hearts was not changed. Nkx2-5 mutant embryonic hearts showed defective coronary vessel organization, which was improved by intermittent mild hyperoxia. The results of our study showed that mild gestational hyperoxia therapy rescued genetic cardiac malformation induced by Nkx2-5 mutation in part.

Background: Vasculature is formed by responding to homeostatic tissue demands including in developing hearts. Hypoxia generally stimulates vascular formation in which vascular endothelial growth factor A (VEGF-A) plays a critical role. Gestational hypoxia increases the risk of low intrauterine growth and low birth weight, both of which are known to increase the risk of the fetus developing cardiovascular defects. In fact, continuous gestational mild hypoxia (14% O 2 ) from the mid-embryonic stage causes cardiac anomalies accompanied by a thinning compact layer in mice in vivo. Because coronary vasculature formation is necessary for compact layers to thicken, we hypothesized that defective coronary vessel organization is related to the thinning compact layer under gestational hypoxia conditions. Results: Continuous gestational mild hypoxia (14% O 2 ) applied from embryonic day 10.5 (E10.5) reduced the expression of VEGF-A mRNA and proteins by over 60% in E12.5 hearts relative to control normoxic hearts. Formation of CD31-positive vascular plexus, blood islands, and microvessels in embryonic ventricles were stunted by gestational hypoxia compared to control E12.5 hearts. Conclusions: Our results suggest that mild hypoxia (14% O 2 ) does not induce coronary vessel organization or VEGF-A expression in developing mouse hearts, opposing the general effects of hypoxia-triggering vascular organization and VEGF-A expression. K E Y W O R D Scoronary vessel, heart, hypoxia, VEGF-A