Reperfusion-activated Akt kinase prevents apoptosis in transgenic mouse hearts overexpressing insulin-like growth factor-1.

Abstract -Hearts of wild-type and insulin-like growth factor-1 overexpressing (Igf-1(+/-)) transgenic mice were subjected to Langendorff perfusions and progressive periods of ischemia followed by reperfusion. Apoptosis was measured by DNA nucleosomal cleavage and a hairpin probe labeling assay to detect single-base overhang. Transgenic hearts subjected to 20 minutes of ischemia and 4 hours of reperfusion (I/R) sustained a rate of apoptosis of 1.8+/-0.3% compared with 4.6+/-1.1% for wild-type controls (n=4; P<0.03). Phosphorylation of the protein kinase Akt/protein kinase B was elevated 6.2-fold in transgenic hearts at baseline and increased another 4.4-fold within 10 minutes of reperfusion, remaining elevated for up to 2 hours. I/R activated Akt in wild-type hearts but to a lesser extent (1.6+/-0.3-fold). Pretreatment of transgenic hearts with wortmannin immediately before and during ischemia eliminated reperfusion-mediated activation of Akt and neutralized the resistance to apoptosis. The stress-activated kinase p38 was also activated during ischemia and reperfusion in both wild-type and transgenic hearts. Perfusion with the p38 inhibitor SB203580 (10 micromol/L) blocked both p38 activation and phosphorylation of Akt and differentially modulated apoptosis in wild-type and transgenic hearts. Pretreatment with SB203580 reduced apoptosis in wild-type hearts but increased apoptosis in transgenic hearts. These results demonstrate that Akt phosphorylation during I/R is modulated by IGF-1 and prevents apoptosis in hearts that overexpress the IGF-1 transgene.

Molecular regulation of the endothelin-1 gene by hypoxia. Contributions of hypoxia-inducible factor-1, activator protein-1, GATA-2, AND p300/CBP.

Endothelin-1 (ET-1) is a peptide hormone with potent vasoconstrictor properties which is synthesized and secreted predominantly by vascular endothelial cells. Its production is regulated by numerous stimuli including ischemia and hypoxia, and the enhanced levels that occur during myocardial ischemia may contribute to the progression of heart failure. We reported previously a preliminary characterization of a hypoxia-inducible factor-1 (HIF-1) binding site in the human ET-1 promoter which contributed to the activation of ET-1 expression in endothelial cells. We report here that the HIF-1 binding site alone is not sufficient for the response to hypoxia but requires an additional 50 base pairs of flanking sequence that includes binding sites for the factors activator protein-1 (AP-1), GATA-2, and CAAT-binding factor (NF-1). Mutation of any one of these sites or the HIF-1 site eliminated induction by hypoxia. Mutations of the AP-1 and GATA-2 sites, but not the HIF-1 site, were complemented by overexpressing AP-1, GATA-2, HIF-1alpha, or the activator protein p300/CBP, restoring the response to hypoxia. Binding studies in vitro confirmed physical associations among GATA-2, AP-1, and HIF-1 factors. Overexpression or depletion of p300/CBP modulated the level of ET-1 promoter expression as well as the endogenous ET-1 transcript but did not change the fold induction by hypoxia in either case. Regulation of the ET-1 promoter by hypoxia in non-endothelial cells required overexpression of GATA-2 and HIF-1alpha. The results support essential roles for AP-1, GATA-2, and NF-1 in stabilizing the binding of HIF-1 and promoting recruitment of p300/CBP to the ET-1 hypoxia response complex.

Rapid activation of neutral sphingomyelinase by hypoxia-reoxygenation of cardiac myocytes.

Elevated levels of oxygen free radicals have been implicated in the pathways of reperfusion injury to myocardial tissue. The targets for free radicals may include specific as well as random intracellular components, and part of the cellular response is the induction of extracellularly activated and stress-activated kinases. The intermediate signals that initiate these stress responses are not known. Here we show that one of the earliest responses of cardiac myocytes to hypoxia and reoxygenation is the activation of neutral sphingomyelinase and accumulation of ceramide. Ceramide increased abruptly after reoxygenation, peaking at 10 minutes with 225+/-40% of the control level. Neutral sphingomyelinase activity was induced with similar kinetics, and both activities remained elevated for several hours. c-Jun N-terminal kinase (JNK) was also activated within the same time frame. Treatment of cardiac myocytes with extracellular ceramides also activated JNK. Pretreating cells with antioxidants quenched sphingomyelinase activation, ceramide accumulation, and JNK activation. Ceramide did not accumulate in reoxygenated nonmuscle fibroblasts, and JNK was not activated by reoxygenation in these cells. The results identify neutral sphingomyelinase activation as one of the earliest responses of cardiac myocytes to the redox stress imposed by hypoxia-reoxygenation. The results are consistent with a pathway of ceramide-mediated activation of JNK.

Hypoxia-activated apoptosis of cardiac myocytes requires reoxygenation or a pH shift and is independent of p53.

Ischemia and reperfusion activate cardiac myocyte apoptosis, which may be an important feature in the progression of ischemic heart disease. The relative contributions of ischemia and reperfusion to apoptotic signal transduction have not been established. We report here that severe chronic hypoxia alone does not cause apoptosis of cardiac myocytes in culture. When rapidly contracting cardiac myocytes were exposed to chronic hypoxia, apoptosis occurred only when there was a decrease in extracellular pH ([pH](o)). Apoptosis did not occur when [pH](o) was neutralized. Addition of acidic medium from hypoxic cultures or exogenous lactic acid stimulated apoptosis in aerobic myocytes. Hypoxia-acidosis-mediated cell death was independent of p53: equivalent apoptosis occurred in cardiac myocytes isolated from wild-type and p53 knockout mice, and hypoxia caused no detectable change in p53 abundance or p53-dependent transcription. Reoxygenation of hypoxic cardiac myocytes induced apoptosis in 25-30% of the cells and was also independent of p53 by the same criteria. Finally, equivalent levels of apoptosis, as demonstrated by DNA fragmentation, were induced by ischemia-reperfusion, but not by ischemia alone, of Langendorff-perfused hearts from wild-type and p53 knockout mice. We conclude that acidosis, reoxygenation, and reperfusion, but not hypoxia (or ischemia) alone, are strong stimuli for programmed cell death that is substantially independent of p53.

Activation of metallothionein gene expression by hypoxia involves metal response elements and metal transcription factor-1.

Metallothioneins (MTs) are a family of stress-induced proteins with diverse physiological functions, including protection against metal toxicity and oxidants. They may also contribute to the regulation of cellular proliferation, apoptosis, and malignant progression. We reported previously that the human (h)MT-IIA isoform is induced in carcinoma cells (A431, SiHa, and HT29) exposed to low oxygen, conditions commonly found in solid tumors. The present study demonstrates that the genes for hMT-IIA and mouse (m)MT-I are transcriptionally activated by hypoxia through metal response elements (MREs) in their proximal promoter regions. These elements bind metal transcription factor-1 (MTF-1). Deletion and mutational analyses of the hMT-IIA promoter indicated that the hMRE-a element is essential for basal promoter activity and for induction by hypoxia, but that other elements contribute to the full transcriptional response. Functional studies of the mMT-I promoter demonstrated that at least two other MREs (mMRE-d and mMRE-c) are responsive to hypoxia. Multiple copies of either hMRE-a or mMRE-d conferred hypoxia responsiveness to a minimal MT promoter. Mouse MT-I gene transcripts in fibroblasts with targeted deletions of both MTF-1 alleles (MTF-1(-/-); dko7 cells) were not induced by zinc and showed low responsiveness to hypoxia. A transiently transfected MT promoter was unresponsive to hypoxia or zinc in dko7 cells, but inductions were restored by cotransfecting a mouse MTF-1 expression vector. Electrophoretic mobility shift assays detected a specific protein-DNA complex containing MTF-1 in nuclear extracts from hypoxic cells. Together, these results demonstrate that hypoxia activates MT gene expression through MREs and that this activation involves MTF-1.

Hypoxia regulates beta-enolase and pyruvate kinase-M promoters by modulating Sp1/Sp3 binding to a conserved GC element.

The transcription rates of glycolytic enzyme genes are coordinately induced when cells are exposed to low oxygen tension. This effect has been described in many cell types and is not restricted to species or phyla. In mammalian cells, there are 11 distinct glycolytic enzymes, at least 9 of which are induced by hypoxia. Recent reports described a role for the hypoxia-inducible factor-1 (HIF-1) in the transcriptional activation of lactate dehydrogenase A, aldolase-A, phosphoglycerate kinase, and enolase-1 genes. It is not known whether the HIF-1 factor acts exclusively to regulate these genes during hypoxia, or how the other genes of the pathway are regulated. In this paper, we describe analyses of the muscle-specific pyruvate kinase-M and beta-enolase promoters that implicate additional mechanisms for the regulation of glycolytic enzyme gene transcription by hypoxia. Transient transcription of a reporter gene directed by either promoter was activated when transfected muscle cells were exposed to hypoxia. Neither of these promoters contain HIF-1 binding sites. Instead, the hypoxia response was localized to a conserved GC-rich element positioned immediately upstream of a GATAA site in the proximal promoter regions of both genes. The GC element was essential for both basal and hypoxia-induced expression and bound the transcription factors Sp1 and Sp3. Hypoxia caused the progressive depletion of Sp3 determined by DNA binding studies and Western analyses, whereas Sp1 protein levels remained unchanged. Overexpression of Sp3 repressed expression of beta-enolase promoters. It is concluded that hypoxia activates these glycolytic enzyme gene promoters by down-regulating Sp3, thereby removing the associated transcriptional repression.

Hypoxia regulates expression of the endothelin-1 gene through a proximal hypoxia-inducible factor-1 binding site on the antisense strand.

Endothelin-1 (ET-1) is a peptide hormone with potent vasoconstrictor properties that is synthesized and secreted predominantly by vascular endothelial cells. Its production is regulated by numerous stimuli including ischemia and hypoxia, and the enhanced levels that occur during myocardial ischemia may contribute to the progression of heart failure. We previously reported that ET-1 expression was induced by both hypoxia and transition metals in endothelial cells (ECs). Here we define an element in the proximal promoter of the ET-1 gene that is responsible for this induction. By using deletions and site directed mutagenesis of the human ET-1 promoter, in combination with electrophoretic gel mobility shifts and transient expression assays in human ECs, we identified an active hypoxia-inducible factor 1 (HIF-1) binding site starting at position -118 upstream of the transcription start site on the non-coding DNA strand. Mutation of this site eliminated induction by hypoxia without affecting basal (aerobic) expression, and the mutated sequence did not display hypoxia-specific binding of HIF-1.

Regulated expression of a foreign gene targeted to the ischaemic myocardium.

OBJECTIVES: Regulated expression of transferred foreign genes may be an important feature of gene therapy. Because coronary artery disease often involves intermittent myocardial ischaemia followed by periods of normal cardiac function it will probably be necessary to regulate the expression of putative therapeutic/cardioprotective genes directly in response to ischaemia-associated signals. The objectives of the current study were to develop a combination of gene regulatory components that can be used to target a product to the myocardium and limit the expression of the gene to periods of ischaemic activity. METHODS: Expression plasmids were constructed containing muscle-specific promoters and hypoxia-responsive enhancer elements linked to a reporter gene. The regulation of these constructs by hypoxia or experimental ischaemia was measured following transient expression in cultured cells or after direct injection of DNA into the rabbit myocardium. RESULTS: A single set of hypoxia response elements placed immediately upstream of the minimal muscle-specific alpha-myosin heavy chain promoter conferred potent positive regulation of this promoter by hypoxia in vitro and by ischaemia in vivo. Induction by ischaemia persisted for at least 4 h and returned to the baseline level within 8 h. CONCLUSIONS: Hypoxia responsive regulatory elements, in combination with weak tissue-restricted promoters incorporated into an appropriate vector system may allow controlled expression of a therapeutic gene in ischaemic myocardium.

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.

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

OBJECTIVES: Endothelin-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. METHODS: ET-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. RESULTS: The 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. CONCLUSIONS: Et-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.

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.

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)

Induction and nuclear accumulation of fos and jun proto-oncogenes in hypoxic cardiac myocytes.

Hypoxic and ischemic stresses cause a series of well documented changes in myocardial cells and tissues, including increased anaerobic glycolysis, loss of contractility, changes in lipid and fatty acid metabolism, and eventual irreversible membrane damage and cell death. In this article we describe changes in the expression and regulation of the proto-oncogenes fos and jun in cardiac myocytes exposed to severe hypoxia. The mRNAs encoding c-Fos, c-Jun, Jun-D, and Jun-B were induced within 1 h of exposure to hypoxia, increased 5-10-fold between 1 and 4 h and then declined. These inductions coincided with loss in myocyte contractility but occurred before there was irreversible cell damage or significant ATP loss. Immunostaining with anti-Fos and anti-Jun antibodies revealed the accumulation of these proteins in hypoxic cell nuclei. Pre-treatment of cells with protein kinase inhibitors significantly repressed the response at the mRNA level. We propose that hypoxic stress in these cells activates signal transduction pathways, possibly involving protein kinases, that result in the inductions of fos and jun gene families. Therefore AP1 may regulate myocardial adaptive responses to hypoxia in advance of energy depletion, cell damage, or reoxygenation.

Negative lusitropy and abnormal calcium handling in hypoxic cardiac myocytes exposed to the calcium-sensitizer EMD 53998.

Positive inotropic agents that increase the sensitivity of myofilaments to calcium have recently been described (Kitada et al., 1987; Cottney et al., 1990; Ferroni et al., 1991; Lee and Allen, 1991; Beier et al., 1992). These drugs appear to augment contractility independently of cAMP or calcium, and thus may have fewer of the adverse side effects seen with other currently available agents (Katz, 1986; Packer 1989). The clinical utility of "calcium-sensitizers" has been questioned on the theoretical grounds that such agents may interfere with relaxation and impair diastolic function (Hajjar and Gwathmey, 1991). Previous studies have shown a small but significant negative lusitropic effect of the calcium sensitizer EMD 53998 in ferret papillary muscle, although this effect was considered to be outweighed by powerful augmentation of contractility. Modelling studies have suggested that the impairment of relaxation by calcium-sensitizers may be even more severe when myocardial calcium is abnormally elevated, such as in hypoxia (Allen and Orchard, 1987; Lodge and Gelband, 1988) and end-stage heart failure (Hajjar and Gwathmey, 1991). We have examined the effects of EMD 53998 and milrinone on contractility and calcium flux in a cell culture model of myocardial hypoxia. The results indicate that increased calcium sensitivity results in marked impairment of relaxation under hypoxic conditions, possibly due to the impaired calcium sequestration and increased calcium availability exhibited by hypoxic myocytes. These studies show that the effects of calcium sensitizers can be strongly influenced by the prevailing status of intracellular calcium handling, and may be deleterious in the diseased or ischemic myocardium.