The relationship between hypoxia and regulation of nitric oxide synthase (NOS) in myocardial tissue is not well understood. We investigated the role of hypoxia inducible factor-1 (HIF-1) on expression of the inducible NOS (iNOS) in myocardial cells in vivo and in vitro. In situ hybridization in myocardial tissue from rats exposed to hypoxia for 3 weeks demonstrated increased iNOS mRNA expression. Northern analysis of RNA from hearts of those animals and from cells exposed to hypoxia for 12 hours in vitro demonstrated an increase of HIF-1 RNA expression. Electrophoretic mobility shift assays using 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 using the murine iNOS promoter resulted in a 3.43-fold increase in promoter activity under hypoxia compared with normoxia. Mutation or deletion of the HIF-1 site eliminated the hypoxic response. As cytokines have been shown to regulate iNOS expression in myocardial cells, cultured neonatal cardiac myocytes were stimulated with interleukin-1beta causing a dramatic induction of iNOS protein expression under normoxia, with further augmentation under hypoxia. Transient transfection of cells stimulated with interleukin-1beta showed an increased iNOS promoter activity under normoxic conditions compared with unstimulated cells, with a further increase in response to hypoxia, which was dependent on HIF-1. These results demonstrate that hypoxia causes an increase in iNOS expression in cardiac myocytes and that HIF-1 is essential for the hypoxic regulation of iNOS gene expression.

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The slow/cardiac troponin C (cTnC) gene has been used as a model system for defining the molecular mechanisms that regulate cardiac and skeletal muscle-specific gene expression during mammalian development. cTnC is expressed continuously in both embryonic and adult cardiac myocytes but is expressed only transiently in embryonic fast skeletal myotubes. We have reported previously that cTnC gene expression in skeletal myotubes is controlled by a developmentally regulated, skeletal muscle-specific transcriptional enhancer located within the first intron of the gene (bp 997 to 1141). In this report, we show that cTnC gene expression in cardiac myocytes both in vitro and in vivo is regulated by a distinct and independent transcriptional promoter and enhancer located within the immediate 5' flanking region of the gene (bp -124 to +32). DNase I footprint and electrophoretic mobility shift assay analyses demonstrated that this cardiac-specific promoter/enhancer contains five nuclear protein binding sites (designated CEF1, CEF-2, and CPF1-3), four of which bind novel cardiac-specific nuclear protein complexes. Functional analysis of the cardiac-specific cTnC enhancer revealed that mutation of either the CEF-1 or CEF-2 nuclear protein binding site abolished the activity of the cTnC enhancer in cardiac myocytes. Taken together, these results define a novel mechanism for developmentally regulating a single gene in multiple muscle cell lineages. In addition, they identify previously undefined cardiac-specific transcriptional regulatory motifs and trans-acting factors.

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The slow/cardiac troponin C (cTnC) gene is expressed in three distinct striated muscle lineages: cardiac myocytes, embryonic fast skeletal myotubes, and adult slow skeletal myocytes. We have reported previously that cTnC gene expression in cardiac muscle is regulated by a cardiac-specific promoter/enhancer located in the 5' flanking region of the gene (bp -124 to +1). In this report, we demonstrate that the cTnC gene contains a second distinct and independent transcriptional enhancer which is located in the first intron. This second enhancer is skeletal myotube specific and is developmentally up-regulated during the differentiation of myoblasts to myotubes. This enhancer contains three functionally important nuclear protein binding sites: a CACCC box, a MEF-2 binding site, and a previously undescribed nuclear protein binding site, designated MEF-3, which is also present in a large number of skeletal muscle-specific transcriptional enhancers. Unlike most skeletal muscle-specific transcriptional regulatory elements, the cTnC enhancer does not contain a consensus binding site (CANNTG) The expression of many muscle-specific proteins is developmentally regulated at the level of transcription (7,34,39,66,85). Thus, one approach to understanding the molecular basis of mammalian myogenesis is to elucidate the transcriptional mechanisms that regulate the expression of muscle-specific genes. The identification and characterization of the basic helix-loop-helix (bHLH) family of muscle-determining transcription factors, including MyoD, myogenin, myf-5, and MRF4/herculin/myf-6, has added significantly to our understanding of skeletal myogenesis (1,3,10,17,49,61,80). Expression of each of these transcription factors appears to be sufficient to activate the skeletal muscle phenotype in many types of cultured cells (77), and hexanucleotide binding sites for these factors (CANNTG), termed E boxes, have been identified in most but not all skeletal muscle-specific transcriptional regulatory elements studied to date (reviewed in reference 71). bHLH proteins each contain a basic domain which is required for DNA binding and an HLH region which is involved in the formation of homo-and heterodimers (6,9,74). The basic domain of each myogenic bHLH family member contains conserved alanine and threonine residues that distinguish them from the nonmyogenic bHLH transcription factors (5, 9).

These results define a link between cell proliferation and programmed cell death in VSMC via the same signal transduction pathway, namely activation of the serine/threonine kinase Akt, which may have significant implication for the development of vascular diseases or remodeling.

Human cytochrome P450s 2C9 and 2C19 metabolize many important drugs including tolbutamide, phenytoin, and (S)-warfarin. Although they differ at only 43 of 490 amino acids, sulfaphenazole (SFZ) is a potent and selective inhibitor of P450 2C9 with an IC50 and a spectrally determined binding constant, KS, of <1 microM. P450 2C19 is not affected by SFZ at concentrations up to 100 microM. A panel of CYP2C9/2C19 chimeric proteins was constructed in order to identify the sequence differences that underlie this difference in SFZ binding. Replacement of amino acids 227-338 in 2C19 with the corresponding region of 2C9 resulted in high-affinity SFZ binding (KS approximately 4 microM) that was not seen when a shorter fragment of 2C9 was substituted (227-282). However, replacement of amino acids 283-338 resulted in extremely low holoenzyme expression levels in Escherichia coli, indicating protein instability. A single mutation, E241K, which homology modeling indicated would restore a favorable charge pair interaction between K241 in helix G and E288 in helix I, led to successful expression of this chimera that exhibited a KS < 10 microM for SFZ. Systematic replacement of the remaining differing amino acids revealed that two amino acid substitutions in 2C19 (N286S, I289N) confer high-affinity SFZ binding (KS < 5 microM). When combined with a third substitution, E241K, the resulting 2C19 triple mutant exhibited a high cataltyic efficiency for warfarin metabolism with the relaxed stereo- and regiospecificity of 2C19 and a lower KM for (S)-warfarin metabolism (<10 microM) typical of 2C9.

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