Keith A. Webster

Hypoxia/Reoxygenation Stimulates Jun Kinase Activity Through Redox Signaling in Cardiac Myocytes

Hypoxia and reoxygenation are principal components of myocardial ischemia and reperfusion and have distinctive effects on the tissue. Both conditions have been associated with inflammation, necrosis, apoptosis, and myocardial infarction. Using a cell culture model of ischemia and reperfusion in which cardiac myocytes were exposed to cycles of hypoxia and reoxygenation, we report here that reoxygenation, but not hypoxia alone, caused sustained approximately 10-fold increases in phosphorylation of the amino-terminal domain of the c-jun transcription factor. The activation was similar to treatments with anisomycin or okadaic acid and correlated with the hypoxia-mediated depression of intracellular glutathione. Reoxygenation-induced c-Jun kinase activity was reduced by preincubating myocytes during the hypoxia phase with the spin-trap agent alpha-phenyl N-tert-butylnitrone or with N-acetylcysteine. The kinase activation was also inhibited by the tyrosine kinase inhibitor genistein but not by other protein kinase inhibitors. These results implicate unquenched reactive oxygen intermediates as the stimulus that initiates a kinase pathway involving the stress-activated protein kinases (JNKs/SAPKs) in reoxygenated cardiac myocytes.

Physical and functional sensitivity of zinc finger transcription factors to redox change.

Redox regulation of DNA-binding proteins through the reversible oxidation of key cysteine sulfhydryl groups has been demonstrated to occur in vitro for a range of transcription factors. The direct redox regulation of DNA binding has not been described in vivo, possibly because most protein thiol groups are strongly buffered against oxidation by the highly reduced intracellular environment mediated by glutathione, thioredoxin, and associated pathways. For this reason, only accessible protein thiol groups with high thiol-disulfide oxidation potentials are likely to be responsive to intracellular redox changes. In this article, we demonstrate that zinc finger DNA-binding proteins, in particular members of the Sp-1 family, appear to contain such redox-sensitive -SH groups. These proteins displayed a higher sensitivity to redox regulation than other redox-responsive factors both in vitro and in vivo. This effect was reflected in the hyperoxidative repression of transcription from promoters with essential Sp-1 binding sites, including the simian virus 40 early region, glycolytic enzyme, and dihydrofolate reductase genes. Promoter analyses implicated the Sp-1 sites in this repression. Non-Sp-1-dependent redox-regulated genes including metallothionein and heme oxygenase were induced by the same hyperoxic stress. The studies demonstrate that cellular redox changes can directly regulate gene expression in vivo by determining the level of occupancy of strategically positioned GC-binding sites.

Regulation of fos and jun immediate-early genes by redox or metabolic stress in cardiac myocytes.

We have previously demonstrated coordinate inductions of c-fos, c-jun, jun B, and jun D in cardiac myocytes exposed to hypoxia for 2 to 4 hours. Induction of these transcripts occurred before any significant loss of intracellular ATP. In the present study, the origin of the signal(s) that regulates immediate-early gene induction was investigated by comparing the effects of hypoxia with those of the metabolic inhibitors cyanide, deoxyglucose and cyanide combined, and iodoacetic acid. Cyanide, an inhibitor of oxidative metabolism, closely mimicked the metabolic effects of hypoxia, with elimination of oxygen consumption, increased lactate production, and minimal decline in ATP levels under both conditions. Compared with hypoxia, cyanide mediated small transient inductions of fos and jun transcripts that followed a different time course. The combination of cyanide and deoxyglucose resulted in inhibition of lactate production as well as respiration, and ATP dropped rapidly to 20% of control levels. The loss of intracellular ATP was followed by fourfold inductions of c-fos and c-jun with minor changes in jun B and jun D transcript levels. Similarly, iodoacetic acid caused a major (90%) loss of ATP and irreversible cell damage as measured by leakage of creatine phosphokinase enzyme and loss of membrane arachidonic acid; ATP loss was followed by fivefold to sevenfold inductions of c-fos, c-jun and jun B transcripts.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardioprotection in an in vitro model of hypoxic preconditioning.

Short periods of myocardial ischemia appear to provide protection against subsequent prolonged ischemic episodes in experimental animals and in man. This phenomenon, known as ischemic preconditioning, has not yet been characterized at the cellular or molecular levels; however, tissue hypoxia appears to be required. In this study, we used a previously developed method for hypoxic cardiac myocyte culture in order to establish a model for ischemic (or hypoxic) preconditioning in cell culture. We demonstrate that cultured neonatal rat cardiac myocytes preconditioned by 25 min of exposure to hypoxia followed by reoxygenation were protected against membrane damage for up to 6 h of prolonged severe hypoxia, as determined by arachidonic acid release and contractile recovery. In contrast, non-preconditioned myocytes exhibited significant hypoxic damage after 2-4 h. Pretreatment of cells with PMA, a tumor-promoting phorbol ester, mimicked the protective effects of hypoxic preconditioning; pretreatment with the muscarinic cholinergic agonist carbachol had no effect. Our data suggests that isolated myocytes in culture remain competent to be preconditioned by hypoxia, through a pathway that may involve the activation of protein kinase C.

Cell-specificity and signaling pathway of endothelin-1 gene regulation by hypoxia

OBJECTIVESnEndothelin-1 (ET-1) is a potent vasoconstrictor that is expressed in endothelial cells and in many other cells and tissues. Increased plasma levels of the peptide have been associated with ischemic heart disease, atherosclerosis, and myocardial infarction. The objectives of the current study were (1) to determine the tissue specificity for induction of the ET-1 gene by hypoxia, (2) to determine whether the hypoxia regulatory pathway is the same as that in other hypoxia regulated genes and (3) to analyze the contributions of protein kinases for basal and induced expression of ET-1.nnnMETHODSnET-1 transcript levels were measured by Northern blot and quantitative polymerase chain reaction in endothelial and non-endothelial cells following exposure to hypoxia. Regulatory steps within the pathway were identified by treating aerobic or hypoxic cultures with cycloheximide, PMA, a series of selective protein kinase inhibitors, and transition metals. The effects on ET-1 transcripts were compared with the ubiquitous hypoxia inducible pyruvate kinase gene.nnnRESULTSnThe induction of ET-1 by hypoxia in vitro occurred exclusively in early passage endothelial cells. This induction was prevented by treatment with the protein synthesis inhibitor cycloheximide and was at least partially mimicked by treatment with transition metals. Induction by hypoxia was not effected by inhibitors of protein kinase C, protein kinase A, calcium-calmodulin dependent protein kinase, or cyclic GMP dependent protein kinase. The basal expression was decreased and hypoxic induction was eliminated by treating cells with tyrosine kinase-selective inhibitors.nnnCONCLUSIONSnEt-1 induction by hypoxia requires endothelial cell-specific factor(s) or steps, new protein synthesis, and may involve a haeme protein-containing pathway in oxygen sensing. A protein tyrosine kinase step is implicated for both basal and induced expression of the ET-1 gene.

Molecular regulation of cardiac myocyte adaptations to chronic hypoxia

The effects of chronic hypoxia on isolated neonatal rat cardiac myocytes were investigated in a model system of myocardial hypoxia. Spontaneously beating myocardiocytes were cultured for up to one week inside an environmental chamber at an oxygen tension of between 4 and 8 mmHg. In order to stimulate a chronic reduced flow condition fresh hypoxic culture medium was replenished frequently to eliminate or minimize contributions of extracellular metabolite build-up, pH changes, or energy depletion. Under these conditions contractions became progressively impaired and irregular compared with aerobic cultures and beating frequency decreased to about 50% of control over 3 days. Reduced contractility was paralleled by a progressive decrease in the basal intracellular level of cAMP. Both of these effects could be reversed by introducing isoproterenol. Visualization of calcium fluxes using the fluorescent calcium chelator Indo-1 demonstrated that the slower contractions were associated with a pronounced decrease in the rate of calcium efflux during muscle relaxation. Changes in the expression of cAMP dependent genes was apparent in the hypoxic cells and the chronic administration of cAMP elevating drugs was toxic specifically to cells under hypoxia. We propose that cAMP may regulate some short and long-term adaptations of cardiac myocytes to chronic hypoxia.

The anti-cancer agent distamycin A displaces essential transcription factors and selectively inhibits myogenic differentiation

The anticancer drug, distamycin A, alters DNA conformation by binding to A/T-rich domains. We propose that binding of the drug to DNA alters transcription factor interactions and that this may alter genetic regulation. We have analyzed the effects of distamycin A upon expression of the muscle-specific cardiac and skeletal α-actin genes which have A/T-rich regulatory elements in their promoters. Distamycin A specifically inhibited endogenous muscle genes in the myogenic C2 cell line and effectively eliminated the myogenic program. Conversely, when 10T1/2C18 derived pleuripotential TA1 cells were induced to differentiate in the presence of distamycin A, adipocyte differentiation was enhanced whereas the numbers of cells committing to the myogenic program decreased dramatically. Using the mobility shift assay, distamycin A selectively inhibited binding of two important transcription factors, SRF and MEF2, to their respective A/T-rich elements. The binding of factors Sp1 and MyoD wer ffected. The inhibition of factor binding correlated with a repression of muscle-specific promoter activity as assayed by transient transfection assays. Co-expression of the myoD gene, driven by a distamycin A-insensitive promoter, failed to relieve the inhibition of these muscle-specific promoters by distamycin A. Additionally, SRF and MEF2 dependent promoters were selectively down regulated by distamycin A. These results suggest that distamycin A may inhibit muscle-specific gene expression by selectively interfering with transcription factor interactions and demonstrate the importance of these A/T-rich elements in regulating differentiation of this specific cell type.