Differential expression of endothelial nitric oxide synthase in coronary and cardiac tissue in hypoxic fetal guinea pig hearts.

OBJECTIVE: The purpose of the present study was to quantify the effect of chronic hypoxia on endothelial nitric oxide synthase (eNOS) gene and protein expression of fetal coronary artery segments and cardiac tissue of fetal guinea pig hearts.

METHODS: Time-mated pregnant guinea pigs (term = 65 days) were housed in room air (NMX, n = 6) or in a hypoxic chamber containing 10.5% O2 for 14 days (HPX14, n = 6). At near term (60 days gestation), fetuses were excised from anesthetized animals via hysterotomy and hearts were removed and weighed. Both coronary artery segments and cardiac ventricle were excised from the same hearts, frozen, and stored at -80 C until ready for study. eNOS mRNA was quantified using real-time polymerase chain reaction (PCR) based on SYBR Green I labeling (BioRad Laboratories, Hercules, CA) using eNOS primers obtained from GeneBank normalized to 18S. eNOS proteins were quantified by Western immunoblotting using eNOS antibody (1:200) and normalized to normoxic controls. eNOS cell-specific localization in the fetal guinea pig heart was performed by double immunofluorescence staining.

RESULTS: Both coronary artery endothelial cells (EC) and cardiomyocytes (CM) but not vascular smooth muscle cells of normoxic hearts exhibited positive immunostaining of eNOS protein. Chronic hypoxia significantly (P < .05) increased both eNOS mRNA and protein levels of coronary artery segments (by 210.6% and 51.4%, respectively) but decreased (P < .05) mRNA and protein of cardiac tissue (by 50.0% and 40.6%, respectively) in the same hearts.

CONCLUSIONS: Chronic fetal hypoxia, after 14 days, induces sustained changes in eNOS gene and eNOS protein expression that differ between coronary and cardiac tissue in the fetal guinea pig heart. This study suggests that while the functional roles of altered eNOS expression in hypoxic fetal hearts remain unclear, the site at which eNOS expression is altered may be important in the adaptive response of the fetal heart to hypoxia.