Identification of a cis-acting regulatory element conferring inducibility of the atrial natriuretic factor gene in acute pressure overload.

To identify the cis-acting regulatory element(s) which control the induction of the atrial natriuretic factor (ANF) gene in acute pressure overload, DNA constructs consisting of promoter elements linked to a reporter gene were injected into the myocardium of dogs, which underwent aortic banding or were sham-operated. Expression of a reporter gene construct harboring the ANF promoter (-3400ANF) was induced 6-12-fold after 7 d of pressure overload. An internal deletion of 556 bp (nucleotide sequence -693 to -137) completely abrogated the inducibility of the ANF reporter gene construct. An activator protein-1 (AP1)-like site (-496 to -489) and a cAMP regulatory element (CRE) (-602 to -596) are located within the deleted sequence. Site-directed mutagenesis of the AP1-like site but not the CRE completely prevented the induction of this construct to acute pressure overload. Further, the AP1-like site was able to confer inducibility of a heterologous promoter (beta-myosin heavy chain) to higher values than controls. Gel mobility shift assay (GMSA) supershift analysis was performed using a radiolabeled probe of the ANF promoter (-506/-483) that included the AP1-like site (ATGAATCA) sequence, as well as a probe converted to contain an AP1 consensus sequence (ATGACTCA). GMSA analysis demonstrated that the ANF AP1-like element could bind both a constitutively expressed factor and the AP1 proteins, and conversion to a true AP1 site increased its affinity for AP1. However, 7 d after the onset of pressure overload, the AP1 proteins were present only at low levels, and the major complex formed by the ANF AP1-like probe was not supershifted by a jun antibody. Using a large animal model of pressure overload, we have demonstrated that a unique cis-acting element was primarily responsible for the overload induction of the ANF gene.

Mild cerebral ischemia induces loss of cyclin-dependent kinase inhibitors and activation of cell cycle machinery before delayed neuronal cell death.

After mild ischemic insults, many neurons undergo delayed neuronal death. Aberrant activation of the cell cycle machinery is thought to contribute to apoptosis in various conditions including ischemia. We demonstrate that loss of endogenous cyclin-dependent kinase (Cdk) inhibitor p16(INK4a) is an early and reliable indicator of delayed neuronal death in striatal neurons after mild cerebral ischemia in vivo. Loss of p27(Kip1), another Cdk inhibitor, precedes cell death in neocortical neurons subjected to oxygen-glucose deprivation in vitro. The loss of Cdk inhibitors is followed by upregulation of cyclin D1, activation of Cdk2, and subsequent cytoskeletal disintegration. Most neurons undergo cell death before entering S-phase, albeit a small number ( approximately 1%) do progress to the S-phase before their death. Treatment with Cdk inhibitors significantly reduces cell death in vitro. These results show that alteration of cell cycle regulatory mechanisms is a prelude to delayed neuronal death in focal cerebral ischemia and that pharmacological interventions aimed at neuroprotection may be usefully directed at cell cycle regulatory mechanisms.

Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis.

BACKGROUND: The importance of free radical homeostasis and apoptosis in normal and diseased hearts and their interrelationships are poorly defined. We tested whether reactive oxygen species can trigger apoptosis in cardiomyocytes, and we explored the underlying pathways. METHODS AND RESULTS: A cell culture model of isolated cardiac cells and different reactive oxygen species (ROS)-generating systems were used. Apoptosis became evident when cardiomyocytes were exposed to either H2O2 or superoxide anion (O2-). Both H2O2- and O2--induced apoptosis of cardiomyocytes were associated with an increase in p53 protein content, whereas protein levels of Bax and Bcl-2 were unaltered. H2O2, but not O2-, induced an increase in the protein content of Bad. Furthermore, H2O2 elicited translocation of Bax and Bad from cytosol to mitochondria, where these factors formed heterodimers with Bcl-2, which was followed by the release of cytochrome c, activation of CPP32, and cleavage of poly(ADP-ribose) polymerase. Interestingly, this pathway was not activated by O2-. Instead, O2- used Mch2alpha to promote the apoptotic pathway, as revealed by the activation of Mch2alpha and the cleavage of its substrate, lamin A. CONCLUSIONS: Taken together, these results indicate that ROS may play an important pathophysiological role in cardiac diseases characterized by apoptotic cell death and suggest that different ROS-induced activations of the apoptotic cell death program in cardiomyocytes involve distinct signaling pathways.

Requirement for protein kinase C in reactive oxygen species-induced apoptosis of vascular smooth muscle cells.

BACKGROUND: Vascular smooth muscle cell (VSMC) apoptosis is a component of a variety of cardiovascular diseases and may be related to reactive oxygen species (ROS). This study was designed to determine the role of protein kinase C (PKC) in ROS-induced VSMC apoptosis. METHODS AND RESULTS: Rat aortic VSMCs were exposed to H(2)O(2), and the nature of cell death was characterized in the absence or presence of different PKC inhibitors. The results demonstrate that exposure of VSMCs to H(2)O(2) led to a dose-dependent (25 to 100 micromol/L) and time-dependent (peak at 2 minutes) activation of PKC. Among the PKC isoforms alpha, beta, delta, epsilon, and zeta, only PKC-alpha and PKC-epsilon were found to change their intracellular distribution on H(2)O(2) treatment. Apoptosis was the predominant form of cell death when PKC had been activated by H(2)O(2) alone or by H(2)O(2) in the presence of 50 nmol/L phorbol 12-myristate 13-acetate. In contrast, necrosis became the predominant form of cell death when PKC had been downregulated by prolonged exposure to 200 nmol/L phorbol 12,13-dibutyrate or inhibited by 50 nmol/L staurosporine, 100 nmol/L calphostin C, or 30 micromol/L H-7. In addition, caspase-3 was activated in H(2)O(2)-induced VSMC apoptosis but not when PKC was downregulated or inhibited. Inhibition of caspase-3 by 50 micromol/L Ac-DEVD-CHO could significantly attenuate H(2)O(2)-induced apoptosis and was not associated with the induction of necrosis. CONCLUSIONS: We conclude that in VSMCs, PKC converts the ROS-induced signals from necrotic cell death to the activation of an apoptotic cell death program. These data imply a novel and important role of PKC for the pathogenesis of such vascular diseases as atherosclerosis, restenosis, and hypertension.

In cardiomyocyte hypoxia, insulin-like growth factor-I-induced antiapoptotic signaling requires phosphatidylinositol-3-OH-kinase-dependent and mitogen-activated protein kinase-dependent activation of the transcription factor cAMP response element-binding protein.

BACKGROUND: A variety of pathologic stimuli lead to apoptosis of cardiomyocytes. Survival factors like insulin-like growth factor-I (IGF-I) exert anti-apoptotic effects in the heart. Yet the underlying signaling pathways are poorly understood. METHODS AND RESULTS: In a model of hypoxia-induced apoptosis of cultured neonatal cardiomyocytes, IGF-I prevented cell death in a dose-dependent manner. Antiapoptotic signals induced by IGF-I are mediated by more than one signaling pathway, because pharmacological inhibition of the phosphatidylinositol-3-OH-kinase (PI3K) or the mitogen-activated protein kinase kinase (MEK1) signaling pathway both antagonize the protective effect of IGF-I in an additive manner. IGF-I-stimulation was followed by a PI3K-dependent phosphorylation of AKT and BAD and an MEK1-dependent phosphorylation of extracellular signal-regulated kinase (ERK) 1 and ERK2. IGF-I also induced phosphorylation of cAMP response element-binding protein (CREB) in a PI3K- and MEK1-dependent manner. Ectopic overexpression of a dominant-negative mutant of CREB abolished the antiapoptotic effect of IGF-I. Protein levels of the antiapoptotic factor bcl-2 increased after longer periods of IGF-I-stimulation, which could be reversed by pharmacological inhibition of PI3K as well as MEK1 and also by overexpression of dominant-negative CREB. CONCLUSIONS: In summary, our data demonstrate that in cardiomyocytes, the antiapoptotic effect of IGF-I requires both PI3K- and MEK1-dependent pathways leading to the activation of the transcription factor CREB, which then induces the expression of the antiapoptotic factor bcl-2.

Reactive oxygen species induce apoptosis of vascular smooth muscle cell.

Apoptosis of vascular smooth muscle cell (VSMC) plays an important role in the genesis of atherosclerosis and restenosis. In order to investigate the role of reactive oxygen species in the induction of VSMC apoptosis, rat VSMCs were treated with glucose oxidase/glucose (GO/G) or diethylmaleate (DEM). The results showed that GO/G and DEM led to VSMC death. Administration of catalase, superoxide dismutase and deferoxamine revealed that H2O2 was the major reactive oxygen species causing cell death, and H2O2O exerted its effect by formation of hydroxyl radical (.OH). GO/G- and DEM-induced VSMC death occurred by apoptosis characterized by "DNA ladders", condensation of nuclei, positive to in situ nick-end labeling and increases in histone-associated DNA fragmentation. This study suggests that H2O2 and its derived form .OH might be related to apoptosis of VSMC in atherosclerosis and restenosis.

Superoxide induces apoptosis in cardiomyocytes, but proliferation and expression of transforming growth factor-beta1 in cardiac fibroblasts.

Cardiomyocyte apoptosis and cardiac fibroblast proliferation are characteristic features of failing myocardium. Here we investigated the effect of superoxide on the cell fate of cardiomyocytes and cardiac fibroblasts. Cultured rat cardiomyocytes or cardiac fibroblasts were treated with superoxide. In response to superoxide stimulation cardiomyocytes underwent apoptosis as revealed by the increase in histone associated DNA fragmentation and positive to in situ nick end-labeling. In contrast, cardiac fibroblasts were stimulated to proliferate as demonstrated by the increase in DNA synthesis detected by [3H]thymidine incorporation and in cell number. Additionally, Northern blot analysis showed that transforming growth factor-beta1, a key factor responsible for myocardial fibrosis, was upregulated in cardiac fibroblasts in response to superoxide stimulation. These data suggest that superoxide can induce such divergent effects as apoptosis in cardiomyocytes and cell growth in cardiac fibroblasts, indicating that it may be a potential factor involved in the pathogenesis of heart failure.

Right atrial dilatation increases inositol-(1,4,5)trisphosphate accumulation. Implications for the control of atrial natriuretic peptide release.

Stretching the right atrium of isolated perfused [3H]inositol-labelled rat hearts was shown to stimulate the phosphatidyl-inositol turnover pathway as demonstrated by the accumulation of [3H]inositol-(1,4,5)trisphosphate and its degradation products. Stimulation was detectable after 1 min with larger increases observed after 10 or 20 min. These findings demonstrate that the myocardium can respond to dilatation by an activation of the phosphatidylinositol turnover pathway. Such a mechanism has implications for the release of atrial natriuretic peptide following right atrial distention.

Ventricular remodeling after acute myocardial infarction.

The term ventricular remodeling has been coined to describe the geometrical changes in size and shape of the left ventricle occurring after large myocardial infarcts. We do not exactly know what initiates this process. Slipping of myofilaments following destruction of connective tissue--probably due to metalloproteinase activation--could be the initial event. As a consequence, wall stress is increased triggering deleterious adaptation processes, such as: - intracardiac angiotensin II generation; - cardiac endothelin formation and release; - pro-apoptotic signals for cardiomyocytes; - hypertrophic signals for fibroblasts and cardiomyocytes. This cascade of events is not only observed in the process of remodeling following myocardial infarction but is also operating during the progression of heart failure. Therapeutic principles therefore are similar in both conditions: - reduction of wall stress (pharmacological or mechanical unloading of the heart); - blockade of angiotensin II generation or of AT1-receptors (ACE-inhibitors or AT1 antagonists); - blockade of endothelin receptors (ET(A)-blockers); - blockade of adrenergic receptors (preferably beta1-adrenergic receptor blockers). Better understanding of the molecular mechanisms of the remodeling process already has fueled the search for new therapeutic interventions (such as endothelin receptor blockers, aldosterone antagonists and growth hormone application). Continuous research in this field may be especially rewarding if we will succeed in identifying the very first step in the cascade.

Endothelin has potent inotropic effects in rat atria.

Endothelin, a novel peptide recently isolated from aortic endothelial cells, is one of the most active vasoconstrictors yet characterized. We report that endothelin is also a very potent inotropic substance, as demonstrated in isolated rat heart atria. The effects were observed at nanomolar concentrations and appeared to be due to a direct action of the peptide on the heart muscle cells. Thus, endothelin is not only a powerful vasoconstrictor but also has potent contractile effects on cardiac myocytes.

Dilatation of the right atrium stimulates phosphatidylinositol turnover.

1. Dilatation of the rat right atria caused a stimulation of the phosphatidylinositol turnover pathway as measured by an increase in the accumulation of inositol phosphates in isolated, perfused hearts. 2. In right atria, increases were detected after 1 min dilatation and maximal increases were observed after 10 min. Dilatation for 10 min caused an increase in inositol monophosphate inositol bisphosphate and inositol trisphosphate from 23.3 +/- 0.9, 15.4 +/- 0.4, and 9.5 +/- 0.3 (mean and s.e.m., n = 7 ct/min per mg tissue) to 74.6 +/- 2.3, 20.2 +/- 1.3 and 13.6 +/- 1.5 (n = 8), respectively (P less than 0.01 for all inositol phosphates.) 3. These results show that the myocardium can respond to dilatation by an activation of the phosphatidylinositol turnover pathway.

Differential effect of hydrogen peroxide and superoxide anion on apoptosis and proliferation of vascular smooth muscle cells.

BACKGROUND: Proliferation and apoptosis of vascular smooth muscle cells (VSMCs) are two important components of atherosclerosis, restenosis, and hypertension. Although reactive oxygen species have been demonstrated to participate in the pathogenesis of these diseases, their precise involvement has not been fully understood. We hypothesized that different reactive oxygen species exert distinct effects on proliferation and apoptosis of VSMCs. METHODS AND RESULTS: Cultured rat VSMCs were exposed to xanthine oxidase/xanthine (XO/X) or H2O2-Fe(II). A single exposure to XO/X predominantly resulted in cell proliferation, whereas frequent exposures to high levels of XO/X predominantly resulted in cell death. Administration of superoxide dismutase and catalase revealed that O2- but not H2O2, was mitogenic to VSMCs, whereas H2O2 was responsible for VSMC death. Treatment with H2O2-Fe(II) alone or in the presence of different hydroxyl radical scavengers showed that VSMC death occurred in a dose-dependent manner and was mediated by the formation of hydroxyl radicals. Cell death caused by XO/X or H2O2-Fe(II) occurred by apoptosis as revealed by condensation of nuclei, appearance of a "DNA ladder," increases in DNA fragmentation, and positive in situ nick-end labeling. Northern blot analysis indicated that bcl-2 and c-fos but not p53 and c-myc may participate in mediating H2O2-Fe(II)-induced VSMC apoptosis. CONCLUSIONS: Different reactive oxygen species exert distinct effects on VSMCs, with O2- inducing proliferation and H2O2 causing apoptosis. Thus, reactive oxygen species might participate in atherosclerosis, restenosis, and hypertension in a dual manner by stimulating proliferation and triggering apoptosis of VSMCs.

p53 regulates mitochondrial membrane potential through reactive oxygen species and induces cytochrome c-independent apoptosis blocked by Bcl-2.

Downstream mediators of p53 in apoptosis induction remain to be elucidated. We report that p53-induced apoptosis occurred in the absence of cytochrome c release into the cytosol. Although Bax was upregulated, it remained largely in the cytosol and there was no detectable translocation to the mitochondria. Bid was not activated as no cleavage could be detected. Thus, the absence of cytochrome c release may be due to the lack of Bax translocation to mitochondria and/or Bid inactivation. Nevertheless, p53-induced apoptosis is still caspase dependent because it could be abolished by z-VAD-fmk. To search for alternative downstream targets of p53, we detected production of reactive oxygen species (ROS) as well as mitochondrial membrane potential (Deltapsi). p53 induced ROS generation, which then caused a transient increase of Deltapsi followed by a decrease. Antioxidants could inhibit the alterations of Deltapsi, thereby preventing apoptosis. z-VAD-fmk was unable to abrogate Deltapsi elevation but inhibited Deltapsi decrease, indicating that Deltapsi elevation and its decrease are two independent events. Bcl-2 may abolish elevation as well as decrease of Deltapsi without interfering with ROS levels. Thus, the ROS-mediated disruption of Deltapsi constitutes a pivotal step in the apoptotic pathway of p53, and this pathway does not involve cytochrome c release.

The background of the MERIT-HF trial.

In the not so distant past, the idea of using beta-blockers as a primary therapy for congestive heart failure to improve symptoms and prognosis seemed paradoxical. The cardiac community reacted with skepticism when, in 1975, the pioneering report of Waagstein et al. appeared in the British Heart Journal. Since then numerous groups have investigated the effects of beta-adrenoceptor antagonists in patients with congestive heart failure. Unfortunately, the results of these trials have sometimes contradicted one another. Exercise tolerance and left ventricular ejection fraction improved in the trials with a duration of treatment of longer than 3 months, but no benefit was observed when beta-blockers were administered for only 1 month. Now, in the year 2000 we have proof for the concept that beta-blockade improves symptoms and prolongs life in heart failure. Three large placebo-controlled clinical trials with more than 9000 patients have shown that carvedilol, bisoprolol and metoprolol significantly reduce morbidity and mortality in heart failure. These agents, therefore, are clearly indicated in the majority of patients with mild to moderate heart failure.

Intracellular superoxide induces apoptosis in VSMCs: role of mitochondrial membrane potential, cytochrome C and caspases.

Apoptosis of vascular smooth muscle cells (VSMCs) is an integral part of cardiovascular diseases including atherosclerosis, hypertension and restenosis. Here we studied the fate of VSMCs in response to intracellular superoxide stimulation. Diethyldithiocarbamic acid (DDC) was used to inhibit copper-zinc superoxide dismutase thereby increasing intracellular superoxide levels. The results show that DDC at a dose from 25-100 micro M is able to induce VSMC apoptosis. Superoxide was found to be responsible for DDC-induced apoptosis. In the apoptotic process mitochondrial membrane potential was decreased and caspase-3, -8 and -9 were activated. Surprisingly, neither cytochrome c release nor Bid cleavage could be observed. These data suggest a role for intracellular superoxide in the regulation of VSMCs apoptosis.

Myocardial stretch stimulates phosphatidylinositol turnover.

The mammalian myocardium responds to stretch by increasing both contractility and the release of atrial natriuretic peptide. These effects are observed in isolated perfused heart preparations as well as in vivo. That atrial natriuretic peptide release can be stimulated by activation of the phosphatidylinositol turnover pathway suggests a possible mechanism by which stretch might activate a biological response. Accordingly, experiments were performed to examine the effect of dilatation of the right atrium on the phosphatidylinositol turnover pathway measured in isolated perfused hearts. Dilatation of the right atrium caused a stimulation of the phosphatidylinositol turnover pathway as measured by an increase in the accumulation of inositol phosphates. In right atria, increases were detected after 1 minute of dilatation, and maximal increases were observed after 10 minutes. Dilatation for 10 minutes caused an increase in inositol monophosphate, inositol bisphosphate, and inositol trisphosphate from 23.3 +/- 0.9, 15.4 +/- 0.4, and 9.5 +/- 0.3 cpm/mg tissue (mean +/- SEM, n = 7) to 74.6 +/- 2.3, 20.2 +/- 1.3, and 13.6 +/- 1.5 cpm/mg tissue (n = 8), respectively (p less than 0.01 for all inositol phosphates). Smaller increases were observed in the other chambers of the hearts. Perfusion with propranolol, prazosin, and atropine (all 1 microM) did not alter the inositol phosphate response to dilatation, indicating that it was not secondary to release of norepinephrine or acetylcholine. Dilatation of the right ventricle also caused a stimulation of inositol phosphate accumulation, but this was lower than after dilatation of the right atrium. These results show that the myocardium can respond to dilatation by an activation of the phosphatidylinositol turnover pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

E2F-1 overexpression in cardiomyocytes induces downregulation of p21CIP1 and p27KIP1 and release of active cyclin-dependent kinases in the presence of insulin-like growth factor I.

The heart is a postmitotic organ unable to regenerate after injury. The mechanisms controlling cell cycle arrest in cardiomyocytes are still unknown. Adenoviral delivery of E2F-1 to primary rat cardiomyocytes resulted in an increase in the expression of key cell cycle activators and apoptosis in >90% of the cells. However, insulin-like growth factor I (IGF-I) rescued cardiomyocytes from E2F-1-induced apoptosis. Furthermore, overexpression of E2F-1 in the presence of IGF-I induced the specific downregulation of total p21(CIP1) and p27(KIP1) protein levels and their dissociation from cyclin-dependent kinases (cdks). In contrast, p16(INK4) and p57(KIP2) protein levels and their association with cdks remained unaltered. The dissociation of p21(CIP1) and p27(KIP1) from their cdk complexes correlated well with the activation of cdk2, cdk4, and cdk6 and the release from cell cycle arrest. Under these circumstances, the number of cardiomyocytes in S phase rose from 1.2% to 23%. These results indicate that IGF-I renders cardiomyocytes permissive for cell cycle reentry. Finally, the specific downregulation of p21(CIP1) and p27(KIP1) further suggests their key role in the maintenance of cell cycle arrest in cardiomyocytes.

Effect of NF-kappa B Inhibition on TNF-alpha-induced apoptosis and downstream pathways in cardiomyocytes.

Heart-specific inhibition of survival pathway gp130 was recently shown to sensitize transgenic mice towards stress stimuli, resulting in rapid onset of cardiac dilatation and heart failure. In order to identify further survival pathways we evaluated the role of transcription factor nuclear factor-kappa B (NF-kappa B) in tumour necrosis factor-alpha (TNF-alpha)-induced apoptosis of cardiomyocytes. TNF-alpha stimulation (10 ng/ml) of both H9c2 cells and primary cardiomyocytes isolated from neonatal Wistar rats resulted in rapid nuclear translocation of NF-kappa B complexes. The NF-kappa B complexes consisted of rel-proteins p50 and p65, as revealed by supershift analysis. Addition of proteasome inhibitor MG132 or adenoviral expression of a truncated I kappa B alpha (I kappa B Delta N) inhibited TNF-alpha-induced NF-kappa B nuclear translocation in a dose-dependent manner. Both neonatal cardiomyocytes and H9c2 cells were resistant to TNF-induced apoptosis. However, specific inhibition of NF-kappa B activation by Ad5-I kappa B alpha Delta N (MOI=50) or MG132 (5 microm) increased apoptosis as measured by subG1-assay (H9c2 cells) and annexin V binding/propidium iodide (neonatal cardiomyocytes, FACS-analysis: 7+/-2% to 26+/-5% annexin V positive/PI negative), respectively. TUNEL-assay double-stained with anti-alpha-sarcomeric actin confirmed apoptosis of neonatal cardiomyocytes. Furthermore, caspase-3 activation was increased by 52+/-7% in neonatal cardiomyocytes after TNF alpha+Ad5-I kappa B alpha Delta N compared to TNF alpha+Ad5-control treatment. Protein levels of hiAP1, hiAP2, x-iAP, bcl-2 and bcl-x(L) were neither downregulated by NF-kappa B inhibition nor upregulated by TNF-alpha stimulation. In summary, cardiomyocytes utilize transcription factor NF-kappa B to activate survival factors in the context of TNF-alpha stimulation. As locally increased levels of TNF-alpha have been detected in heart failure, NF-kappa B activity is essential for cellular homeostasis in the heart.

Angiotensin II and coronary artery disease, congestive heart failure, and sudden cardiac death.

An activated renin-angiotensin system is a major risk factor for cardiovascular events. Angiotensin II acts on AT1 and AT2 receptors. Stimulation of AT1 receptors is associated with endothelial dysfunction, mainly as the consequence of an increased vascular production of superoxide radicals, vasoconstriction, platelet activation, enhanced release of plasminogen activator inhibitor-1, activation of immediate early genes c-fos and c-jun, myocyte hypertrophy, connective tissue formation, endothelin-1 synthesis, and activation of growth factors like PDGF and TGF-beta 1. Stimulation of AT2 receptors can mitigate or abolish the growth promoting effects of AT1 receptor stimulation. The contribution of these effects--single or in combination--on the progression of atherosclerotic lesions, the phenomenon of restenosis and the process of remodeling in heart failure is being progressively elucidated. With increasing knowledge about these relationships the inhibition of AT1 receptors appears as a main target in preventive and reparative strategies in cardiovascular diseases.