Impact of neonatal hyperoxia on the development of the coronary vascular bed: an experimental model for the study of cardiomyopathy associated with premature birth

Abstract Introduction Very preterm birth (PT; <32 weeks of gestation), nearly 2% of all births, leads to short-term complications such as bronchopulmonary dysplasia and retinopathy of prematurity that are driven by compromised organ vascular development. Adults born preterm display an increased risk of ischaemic cardiomyopathy and heart failure. Our group has shown that neonatal rats exposed to hyperoxia, simulating the PT neonatal conditions, develop cardiomyocytes hypertrophy, fibrosis and cardiac dysfunction, or Oxygen-Induced Cardiomyopathy (OIC). Whether cardiac changes also associate with alterations in vascular development in the left ventricle (LV) remains unknown. Purpose We hypothesized that neonatal exposure to hyperoxia impact the development of the LV vascular network. Methods Male pups were exposed to 80% O2 (OIC) or room air (CTRL) from day 3 (P3) to P10 of life. Hearts were collected at P10, 4- and 16 weeks. Arteriolar length and capillary density per cardiomyocyte were measured using stereology (elastin) and immunofluorescence (WGA, α-SMA, CD31). High-resolution 3D reconstruction of the vascular network was achieved by combining fluorescent labelling of coronaries (anti-α-SMA) and capillaries (anti-CD31) with a tissue-cleaning protocol (iDisco+), allowing detailed morphometric characterization of the vascular network. The expression of vascular endothelial growth factors and receptors (VEGFA, VEGFB, VEGFR 1 and 2, NRP1, and PIGF) were quantified by RT-qPCR and Western blots. Differences between groups were analyzed with Student's t-test (P < 0.05) (N = 6 per group). Results At P10, we observed a notable rise in the number of capillaries per cardiomyocyte (0.88 ± 0.05 vs. 0.62 ± 0.02 mm2) and a significant increase in VEGFA (+76%) and NRP1 (+38%) expression in the LV from OIC vs. CTRL rats. By 4 weeks, the OIC group showed a significant reduction in arteriolar length vs. CTRL (6.51 ± 0.76 vs. 9.77 ± 1.12 m/cm3), coupled with an increase in the number of capillaries per cardiomyocyte (1.11 ± 0.05 vs. 0.85 ± 0.03 mm2). VEGFA was decreased (-14%) and PLGF increased (+24%) in the LV of OIC rats. At 16 weeks, the OIC condition displayed a significant increase in the number of capillaries per cardiomyocyte (1.37 ± 0.03 vs. 0.98 ± 0.07 mm2) and a decrease in VEGFA (-23%), along with reduction in its receptors VEGFR1 (-27%), VEGFR2 (-20%) and NRP1 (-17%). Conclusion OIC resulting from neonatal hyperoxia exposure associates with vascular changes in the LV that persist to adulthood. These changes are characterized by a reduction in arteriolar length and an increase in the number of capillaries, accompanied by modulation of the expression of pro-angiogenic VEGFA and receptors. The impact of these vascular alterations on the susceptibility of the LV to ischemic or hypertension-related damage is to be explored. These findings suggest a crucial mechanistic pathway for understanding the risk of cardiac pathologies associated with preterm birth.