Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes

The relationship between hypoxia and regulation of nitric oxide synthase (N OS) in myocardial tissue is not well understood. We investigated the role o f hypoxia inducible factor-1 (HIF-1) on expression of the inducible NOS (iN OS) in myocardial cells in vivo and in vitro, In situ hybridization in myoc ardial tissue from rats exposed to hypoxia for 3 weeks demonstrated increas ed iNOS mRNA expression, Northern analysis of RNA from hearts of those anim als and from cells exposed to hypoxia for 12 hours in vitro demonstrated an increase of HIF-1 RNA expression. Electrophoretic mobility shift assays us ing oligonucleotides containing the iNOS HIF-1 DNA binding site and nuclear extracts from cardiac myocytes showed induction of specific DNA binding in cells subjected to hypoxia, Transient transfection of cardiac myocytes usi ng the murine iNOS promoter resulted in a 3.43-fold increase in promoter ac tivity under hypoxia compared with normoxia, Mutation or deletion of the HI F-1 site eliminated the hypoxic response. As cytokines have been shown to r egulate iNOS expression in myocardial cells, cultured neonatal cardiac myoc ytes were stimulated with interleukin-1 beta causing a dramatic induction o f iNOS protein expression under normoxia, with further augmentation under h ypoxia, Transient transfection of cells stimulated with interleukin-1 beta showed an increased iNOS promoter activity under normoxic conditions compar ed with unstimulated cells, with a further increase in response to hypoxia, which was dependent on HIF-1. These results demonstrate that hypoxia cause s an increase in iNOS expression in cardiac myocytes and that HIF-1 is esse ntial for the hypoxic regulation of iNOS gene expression.