Cardiovascular Research

Cardiovascular Research 1998 39(2):434-441; doi:10.1016/S0008-6363(98)00118-7
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Nuclear factor NF-B in myocardium: developmental expression of subunits and activation by interleukin-1β in cardiac myocytes in vitro

David A.M. Norman, Magdi H. Yacoub and Paul J.R. Barton^*

Cardiothoracic Surgery, Imperial College School of Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK

^* Corresponding author. Tel.: +44 (0)171 351 8184; Fax: +44 (0)171 376 3442; E-mail: p.barton@ic.ac.uk

Received 21 November 1997; accepted 24 February 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: The aims of the study were to investigate the pattern of expression of the major subunits of the NF-B transcription factor complex in human and rat heart development, and to characterise the timing of NF-B activation by interleukin-1β (IL-1β) in rat neonatal cardiac myocytes. Methods: The expression of NF-B subunits p65 and p50 and the inhibitory subunits IB- and IB-β in human and rat myocardial samples was measured by immunoblotting, using antibodies specific to each subunit. The activation of NF-B was measured in neonatal rat cardiac myocytes that were treated with IL-1β for different times (0–60 min). Depletion of the inhibitory factors IB- and IB-β was assessed by immunoblotting. The presence of NF-B DNA binding activity was measured directly in nuclear extracts by electrophoretic mobility shift assay (EMSA). Results: p65, p50, IB- and IB-β are expressed at all stages of development analysed. In human myocardial samples, expression of p50, p65 and IB- show an apparent gradual decline relative to total protein. In contrast, the level of IB-β remained relatively constant, suggesting a significant shift in the ratio of β and subunits with development. In rat myocardium, p65, p50, IB- and IB-β showed a gradual decline during development, with a particularly pronounced decrease between the ten day post-natal and adult samples. Treatment of neonatal rat cardiac myocytes with IL-1β (5 ng/ml) caused a rapid and transient depletion of IB- (reducing to 16±1.6% of initial levels within 5 min, returning to 82±10% within 60 min). A slower, less marked depletion is observed for IB-β (24±6% by 30 min, returning to only 49±5% by 60 min). Rapid and transitory accumulation of NF-B DNA binding activity was detected in the nucleus, with a pattern that correlated with the depletion of IB-. Conclusions: The principal NF-B subunits p65, p50, IB- and IB-β are present throughout development, suggesting that this transcription complex may participate in myocardial gene regulation throughout development and in the adult. Activation by IL-1β demonstrates that NF-B probably plays a direct role in the regulation of gene transcription in response to cytokine activation in cardiac myocytes.

KEYWORDS Development; Cardiac myocyte; NF-B; Interleukin-1β; Rat; Human

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Nuclear factor NF-B is a transcription factor complex originally identified as a regulator of immunoglobulin genes but which has become recognised as being widely, and perhaps ubiquitously, expressed [1–3]. The importance of NF-B in myocardium has been highlighted in a recent study where activation was abrogated using an NF-B-binding decoy oligonucleotide [4], an effect that was shown to prevent myocardial infarction following coronary artery ligation in a rat experimental model. The detailed mechanisms involved in this cardioprotective effect remain to be determined, but specific observations included a decrease in interleukin-6 (IL-6) and VCAM gene expression, which may reduce lymphocyte infiltration. In other studies, direct NF-B activation has been demonstrated in cardiac myocytes in response to IL-1β, [5, 6]hydrogen peroxide [7]and myocardial ischemia [8]. The target genes activated by NF-B in cardiac myocytes remain to be determined. Candidates include the inducible nitric oxide synthase (iNOS) gene, which is upregulated by IL-1β in cardiac myocytes e.g. [9–12]and which contains consensus binding sites that are positively regulated by NF-B in other cell types [13]Similarly, the IL-6 gene promoter is activated by hypoxia in myocytes [14]and in other cells this is mediated by NF-B [15].

The signals that can activate NF-B have been widely studied in many other systems. Positive stimulatory factors include proinflammatory cytokines, bacterial lipopolysaccharides (LPS), oxidants, hypoxia and calcium overload [16–18]. Such factors have clear relevance to clinically important aspects of cardiac myocyte biology, including inflammatory myocardial disease, septic shock, cardiac ischemia and reperfusion injury. A further observation of interest is that NF-B can interact with cardiac myotrophin [19], a protein isolated from hypertrophied hearts of SHR rats and from dilated cardiomyopathic human hearts and that is believed to contribute to the hypertrophic process by stimulating protein synthesis. NF-B may be suppressed by cyclosporin A [20]and participate in the pathway leading to activation of IL-6 by FK506 [21], suggesting that immunosuppression (e.g. in cardiac transplantation) may influence cardiac myocyte NF-B pathways.

In mammalian cells, NF-B is composed of homo- or heterodimers of the rel/NF-B family of proteins. These proteins share a common sequence domain, the Rel-homology domain, which is involved in DNA-binding, nuclear localization and dimerization. Members include p50, p52 (synthesised as p105 and p100 precursors, respectively), p65 (RelA), cRel and RelB, which bind specifically to B sequence motifs located in the promoters of target genes [16]. Of the possible combinations, it is thought that only those that contain p65, c-Rel or RelB function as transcriptional activators, as other subunits do not contain recognised transactivating domains [16]. Most commonly investigated is the p50/p65 heterodimer, which is widely expressed and binds to DNA at the sequence motif (GGGRNNYYCC) [17].

In its inactive form, NF-B is retained in the cytoplasm by inhibitor (IB) molecules, which comprise a distinct family of proteins that bind to NF-B and inhibit nuclear localization and DNA-binding. Inhibition is relieved by a process that involves phosphorylation and ubiquitination of IB, and its subsequent degradation via the proteosome pathway. Five IB isoforms have been described to date. Of these, two (IB- and IB-) are in fact precursors of the p50 and p52 mature subunits [3]. The others, IB-, IB-β and IB-, are separate gene products. IB- is rapidly depleted in many cell types following stimulation. IB-β shows different characteristics in its response time and may differ in its mode of activation [22], leading to speculation that it may play a role in the longer-term responses observed in some cell types [23, 24]. IB-, IB-β and IB- [25, 26]are widely expressed and are probably ubiquitous. One of the genes that NF-B acts upon is the IB- gene. Depletion of IB- following cell stimulation therefore acts to reinitiate its own synthesis via activation of the IB- gene promoter by NF-B. This overall process is rapid such that, following stimulation, IB can be completely degraded within 5 min and subsequently restored to initial levels within 60 min. This feedback loop forms an essential part of the rapid transient response of NF-B.

To date, no study has specifically addressed the expression of subunits in myocardium in adult or at different developmental stages or determined the characteristics of the response of IB- and IB-β following activation. Here, we demonstrate the presence of NF-B subunits p50 and p65, and of the inhibitory subunits IB- and IB-β, in ventricular myocardial samples from fetal, neonatal and adult rat and human heart, as well as in neonatal rat cardiac myocytes that were cultured in vitro. We demonstrate that both IB- and IB-β are subject to rapid transitory degradation following IL-1β stimulation of rat neonatal cardiac myocytes in vitro, with subsequent re-synthesis of IB- within 1 h. There is a concomitant rapid and transitory accumulation of NF-B DNA-binding activity in the nucleus. These data demonstrate that NF-B is present during fetal, neonatal and adult life and that it contributes to the response of cardiac myocytes to cytokine activation.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Materials
Pancreatin was obtained from Gibco. Collagenase was from Worthington Biochemical Corporation. Primaria plates and 60 mm dishes were from Falcon. Cytosine β-D-arabinofuranoside (Ara-C), cycloheximide, DMEM, Medium 199, horse serum (HS), fetal calf serum (FCS), penicillin/streptomycin, glutamine, HEPES, insulin–transferrin–selenium (ITS), phosphate-buffered saline (PBS), phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin and pepstatin were all from Sigma. Human recombinant interleukin-1β (IL-1β) was kindly provided by Dr. Tim Wells, Wellcome–Glaxo. The primary antibodies anti-IB- (sc-371) and anti-p65 (sc-372) were from Santa Cruz and have been widely used in the analysis of these proteins (e.g. [27, 28], respectively). Anti-p50 was a kind gift from Dr. Nancy Rice [29]and anti-desmin was from Sigma (Cat# D8281). In each case, these antibodies revealed a single band of the expected molecular mass, as determined by comparison with standard markers (Rainbow markers, Amersham). Anti-IB-β antibody was a kind gift from Dr. Alain Israel [22]and revealed a single major band of the molecular mass predicted for IB-β with a minor non-specific band of about 66 kDa. Secondary antibodies were from Dako.

2.2 Cell culture
Cardiac myocytes were isolated from the ventricles of one–two-day-old Sprague-Dawley rats by successive digestions with pancreatin (0.6 mg/ml)/collagenase (0.48 mg/ml), according to published protocols [30]. Cardiac myocytes were enriched by selective preplating for 45 min on plastic culture dishes (Primaria), during which time, the majority of non-myocytes adhered to the bottom of the dish. The resulting suspension of cardiac myocytes was plated on pre-gelatinised 60 mm plates at a density of 10⁶ cells per dish in complete medium (DMEM, Medium 199, 10% HS, 5% FCS, 0.1 U/ml penicillin, 0.1 mg/ml streptomycin, HEPES, pH 7.4, 2 mM glutamine). Ara-C was added to the medium to a final concentration of 10^–5 M, to prevent the proliferation of non-myocytes. Following overnight incubation, the medium was changed to either serum-containing (i.e. complete medium as above) or serum-free medium (DMEM, Medium 199, 0.1 U/ml penicillin, 0.1 mg/ml streptomycin, HEPES, pH 7.4, 2 mM glutamine, supplemented with ITS) and incubated for at least 24 h before being used in an experiment. Using this method, cultures containing at least 95% cardiac myocytes were routinely obtained, as determined by FACS analysis using an anti-sarcomeric actin antibody. The investigation was performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986, published by HMSO, London.

2.3 IL-1β stimulation, protein preparation and immunoblot assay
Cardiac myocytes were washed and fed fresh medium before the addition of 5 ng/ml IL-1β at time 0. After 5, 15, 30, 45 and 60 min, cells were washed in ice-cold PBS before being lysed by the addition of 200 µl of lysis buffer (1% SDS, 0.5 mM PMSF, 2.5 µg/ml each of aprotinin and leupeptin, 1 µg/ml pepstatin). Chromosomal DNA was sheared by drawing the sample through a 26.5G needle. The final protein concentration was measured using the BCA Assay (Pierce). Protein (25 µg) was mixed with sample buffer (62.5 mM Tris–HCl, pH 6.8, 10% glycerol, 2% SDS, 0.7 M 2-mercaptoethanol, 0.0025% bromophenol blue), heated to 100°C for 2 min and loaded onto a 10% sodium dodecyl sulphate (SDS)–polyacrylamide gel (25 mM Tris, 192 mM glycine and 1% SDS). Samples were electrophoresed for 3.5 h at 35 mA before being transferred onto Hybond-C super nitrocellulose membranes (Amersham) by Western blotting for 2.5 h at 1 A in 25 mM Tris, 192 mM glycine and 20% methanol. Membranes were immersed in PBS–Tween 20 (PBS-T) and 3% milk protein overnight at 4°C, to block non-specific binding. Primary antibody, diluted 1:1000 (v/v) in PBS-T/3% milk protein, was added to the membranes for 60 min (room temperature); the membranes were rinsed (2x20 min) with PBS-T and subsequently incubated with the appropriate horseradish-peroxidase-conjugated secondary antibody, diluted 1:2000 (v/v) in PBS-T/3% milk protein for 30 min (room temperature). Membranes were rinsed for 2x20 min before the immunoreactive bands were visualised using Amersham ECL reagents, according to the manufacturer's protocol. Immunoreactive bands produced by immunoblot analysis were scanned using Image Analysis 1000 software (Alpha Innotech). Data were from five experiments, each experiment performed in duplicate, and the result were expressed as an average±S.E.M.

2.4 Electrophoretic mobility shift assay (EMSA)
IL-1β stimulation of cardiac myocytes was performed as for total cell lysate before the cells were treated to extract nuclear and cytosolic fractions [31]. After incubation, the cells were washed with ice-cold PBS and detached using trypsin solution (0.5 mg/ml) for 5 min at 37°C. Digestion was stopped by the addition of complete medium and the cells were pelleted by centrifugation for 5 min at 1000 rpm (room temperature). The cell pellet was resuspended in buffer A without Nonidet-P 40 (NP-40) (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF, 2.5 µg/ml each of aprotinin and leupeptin, 1 µg/ml pepstatin). The cells were pelleted by centrifugation for 5 min at 1000 rpm (4°C) and the pellet was resuspended in 200 µl of buffer A with 0.1% NP-40 added. After incubation on ice for 15 min, the suspension was passed through a 26.5G needle ten times and the nuclei were pelleted by centrifugation for 1 min at 12,000 g and 4°C. The supernatant (cytosolic fraction) was transferred to a fresh tube. The nuclear pellet was washed with buffer A twice before lysis by osmotic shock in 40 µl of a solution containing 20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl₂, 0.42 M NaCl, 0.2 mM EDTA, 0.5 mM dithiothreitol (DTT) and 0.5 mM PMSF, 2.5 µg/ml each of aprotinin and leupeptin, and 1 µg/ml pepstatin and then was incubated at 4°C for 30 min with rotation. Nuclear debris was pelleted by centrifugation for 10 min at 4°C and 12,000 g, and the supernatant of nuclear proteins was transferred to a fresh tube. Protein concentrations were determined as for total cell extract.

For EMSA studies, nuclear protein extracts (2 µg) were added to a total volume of 20 µl of reaction mix (40 mM NaCl, 15 mM HEPES, pH 7.9, 1 mM EDTA, 0.5 mM DTT, 10% glycerol and 2.5 µg poly-dIdC) in the presence of 10 fmol of NF-B consensus oligonucleotide cassette, 5'-AGTTGAGGGGACTTTCCCAGGC (Promega), labelled with [³²P]-dATP (Amersham) according to the manufacturer's protocols. These were incubated for 20 min at room temperature before half of the volume was loaded onto a 10% non-denaturing polyacrylamide gel and electrophoresed for 1 h 45 min at 160 V. Supershift analysis was performed by incubating 2 µg of nuclear protein with 1 µg of anti-p65, in a total volume of 20 µl as for the EMSA, for 20 min at room temperature before the addition of the labelled NF-B cassette and a further 20 min incubation. The gel was dried and exposed to film (Kodak) at –70°C between intensifying screens.

2.5 Developmental series
Human fetal and post-natal cardiac muscle was obtained from elective terminations and at autopsy and placed in liquid nitrogen before treatment for Western blot analysis. Adult heart samples were from explanted hearts taken at the time of transplantation. Samples of late fetal (i.e. post 20 weeks) and of post-natal and adult heart were of ventricular muscle only. Earlier samples were predominantly ventricular, although contamination with atrial muscle could not be excluded. Fetal and neonatal samples were judged to be non-pathological and without cardiovascular developmental abnormality. The age of fetal samples was determined by anatomic measurement and is given as the calculated gestational age derived from standard normograms.

Samples of rat (Sprague-Dawley) heart were obtained from fetuses of time-mated females, neonates and adults. For fetal samples, pregnant mothers were sacrificed by cervical dislocation, fetuses were rapidly removed and placed immediately in ice-cold, sterile, calcium-free PBS, pH 7.4. Hearts were dissected and rinsed in ice-cold PBS and were frozen in liquid nitrogen: 12.5 day fetal samples were essentially whole heart, 15 day fetal and older samples were ventricular myocardium (apical 2/3rds). Neonatal samples were obtained by decapitation, the hearts were placed immediately in ice-cold PBS as above, the ventricular myocardium (apical 2/3rds) was removed, rinsed in PBS and frozen in liquid nitrogen. Adult heart samples were obtained by cervical dislocation; the ventricular muscle (apical 2/3rds) was removed, rinsed immediately in ice-cold PBS as above and frozen in liquid nitrogen.

For Western blot analysis, total protein extracts were prepared by homogenising heart tissue or tissue cultured cells in lysis buffer, and the protein content of each sample was determined using the Pierce BCA kit, as for cardiac myocyte cell lysates. A 30-µg amount of protein was prepared, electrophoresed, transferred to membrane and treated with antibodies as previously described.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Expression of NF-B subunits in myocardium
In order to investigate the expression of NF-B subunits in myocardium, total protein extracts were prepared from different stages of human and rat heart development. Equal amounts were analysed by SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis with the commonly found members of the NF-B family; p65, p50, IB- and IB-β. Fig. 1 shows the pattern of expression during human heart development from 12 weeks fetal life to adult. All subunits were expressed at all of the stages of development analysed. The expression of p50 and IB- showed an apparent gradual decline relative to total protein, an effect that may relate to the overall developmental increase in cell mass (see Section 4). In contrast, the level of IB-β remained relatively constant, suggesting a significant shift in the ratio of β and subunits with development. The level of p65 observed in the 17- and 24 week fetal-, nine month post-natal- and adult samples showed an apparent decrease with developmental age, in line with that observed for p50 and IB-. The level detected in the 12- and 33-week fetal samples appeared to be unexpectedly low. Analysis of a second series of immunoblots using the same tissue extracts and antibodies resulted in the same pattern. In the rat heart series (Fig. 2), the abundance of all subunits, including IB-β, showed a gradual decline during development, with a particularly pronounced decrease between the ten day post-natal and adult (60 day) samples.

View larger version (74K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Expression of p65, p50, IB-β and IB- during human heart development. Total cell extracts were prepared from ventricular samples of 12, 17, 24 and 33 week fetal (12w, 17w, 24w and 33w), nine months postnatal (+9m) and adult (Ad) human myocardium. Following SDS–PAGE and immunoblotting, membranes were probed with antibodies for p65, p50, IB-β and IB- subunits and visualised using ECL. Anti-desmin antibody was used as an internal control.

 

View larger version (76K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Expression of p65, p50, IB-β and IB- during rat heart development. Total cell extracts were prepared from ventricular samples of 12, 15, 17 and 19 day embryonic (12ED, 15ED, 17ED and 19ED), neonatal (+0.5D), ten days postnatal (+10D) and adult (Ad) rat myocardium. Following SDS–PAGE and immunoblotting, membranes were probed with antibodies for p65, p50, IB-β and IB- subunits and visualised using ECL. Anti-desmin antibody was used as an internal control.

 
3.2 Stimulation of neonatal cardiac myocytes with IL-1β results in transitory depletion of IB- and IB-β
In order to analyse the activation of NF-B in cardiac myocytes, we tested the ability of a variety of agents to deplete IB-, as assessed by immunoblot analysis. Preliminary experiments confirmed that the addition of IL-1β to cultures of primary rat neonatal cardiac myocytes resulted in IB- depletion after a 5-min incubation (e.g. see Fig. 3) and initial experiments showed that there was no significant difference in magnitude of IB- depletion using IL-1β concentrations ranging from 1 to 100 ng/ml. For all subsequent experiments, 5 ng/ml was used and a prolonged time course at this concentration resulted in the production of iNOS mRNA and protein (within 6 h, reaching a maximum at 24 h), as determined by reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblot analysis (data not shown), in agreement with previous studies [10, 11, 32]. Other preliminary experiments further suggested little difference between the response of cells maintained in defined (serum-free) media compared to those cultured in the presence of serum.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 (A and B) Depletion of IB- and IB-β in rat neonatal cardiac myocytes in response to IL-1β. Cardiac myocytes were incubated for varying times with 5 ng/ml IL-1β before preparation of total cell extracts. Following SDS–PAGE and immunoblotting, membranes were probed using antibodies that were specific for IB- (panel A) or IB-β (panel B) and the resulting complexes visualised by ECL. Each time point was performed in duplicate. Top panels show representative Western blots. Bottom panels show pooled data for five independent experiments. Error bars=S.E.M.

 
The precise timing of IB- depletion was determined in cultures of rat neonatal cardiac myocytes that were stimulated with 5 ng/ml IL-1β for 5, 15, 30, 45 and 60 min. Total cell extracts were prepared and analysed by immunoblot analysis using an IB--specific antibody. As shown in Fig. 3a, the addition of IL-1β resulted in a significant drop in IB- levels within 5 min (16±1.6% of the initial level). Levels remained low for 30 min, but by 60 min, they returned to 82±10% of the initial values. Neonatal rat cardiac myocytes therefore exhibit a transient, reversible depletion of IB- in response to IL-1β, in line with the response noted for other cell types [23, 33]. In order to determine whether the level of IB- detected at 5 and 15 min was due to residual (undegraded) protein or to rapid re-synthesis, cells were treated with IL-1β in the presence of the protein synthesis inhibitor, cycloheximide (Fig. 4). This resulted in a drop in the level of IB- to less than 5% of initial values, indicating that rapid re-synthesis accounts for the major part of the protein level detected at 5 and 15 min.

View larger version (51K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Re-synthesis of IB- and IB-β after depletion in response to IL-1β is prevented by treatment with cycloheximide. Cardiac myocytes from neonatal rats were incubated for varying times with 5 ng/ml IL-1β (I), 5 ng/ml IL-1β+5 µM cycloheximide (I+C) or 5 µM cycloheximide (C) before the preparation of total cell extracts. Following SDS–PAGE and Western blotting, membranes were probed using antibodies that were specific for IB- and IB-β and the resulting complexes were visualised by ECL.

 
The time course of IB-β depletion was followed in the same experimental series using an antibody that was specific for this isoform, as shown in Fig. 3b. Following stimulation, the level fell more slowly than seen for IB-, reaching a minimum by 30 min (24±6% of initial levels). Moreover, after 60 min, only half of the initial observed level (49±5%) was restored. The addition of cycloheximide did not cause any further reduction in the minimum levels of IB-β, suggesting the existence of a significant residual, undepleted pool of IB-β (Fig. 4). Overall, it is apparent from these experiments that the IB-β response differs from that of IB-, being slower to reduce in level (reaching a minimum at 30 min compared to 5–15 minutes for IB-), having a less marked overall reduction (to 24±6% of initial levels compared to 16±1.6%) and being slower to increase following the initial reduction.

3.3 Accumulation of nuclear NF-B DNA-binding activity
As a direct measurement of NF-B activation, we assayed the ability of NF-B to translocate from the cytoplasm into the nucleus in response to IL-1β. Nuclear extracts were prepared from rat cardiac myocytes that were stimulated with IL-1β (5 ng/ml) for the same time intervals as above. As shown in Fig. 5, no DNA binding activity was detected in unstimulated cells. DNA-binding activity was readily detectable within 5 min of the addition of IL-1β, gradually declining over the duration of the 60 min stimulus. On adding anti-p65 antibody, the band was super-shifted, confirming the identity of the complex as NF-B.

View larger version (64K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Transient accumulation of NF-B DNA binding activity in rat neonatal cardiac myocytes in response to IL-1β. Cardiac myocytes were incubated for varying times with 5 ng/ml of IL-1β and nuclear extracts were prepared. An electrophoretic mobility shift assay was performed in the absence (left panel) or presence (right panel) of anti-p65 antibody (Santa Cruz).

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
In this study, we have shown that the major NF-B subunits (p50 and p65) and the NF-B inhibitory subunits, IB- and IB-β, are readily detectable in ventricular myocardial extracts throughout cardiac development and in isolated neonatal cardiac myocytes in culture. Cardiac myocytes occupy over 80% of the volume of the ventricular wall [34]and probably contribute over 90% of protein in whole tissue extracts as used here. We therefore propose that the developmental pattern observed reflects the level of expression in cardiac myocytes. The decrease in abundance of p50 and p65 subunits observed between neonatal and adult samples in both rat and man may be explained in part by the transition from hyperplastic to hypertrophic growth during heart development [35]. This results in a significant increase in cell volume following birth, including deposition of myofibrillar and associated proteins, which may act to mask the effective amounts of p50 and p65 available for translocation to the nucleus. Similarly, the inhibitory molecules IB- and IB-β are present throughout development. In the human heart series, the ratio of IB-β to IB- showed a developmental increase that was not seen in rat.

The activation of NF-B by cytokines and other factors has been widely investigated in a number of cell systems [1, 3]. However, few studies have examined the situation in cardiac myocytes [5–8]. Our data show the activation of NF-B by IL-1β. Stimulation resulted in a rapid and transitory depletion of IB- and a slower, partial, transitory depletion of IB-β. The effects of IL-1β on cardiac myocytes are varied and include direct negative inotropy [36], induction of cardiac hypertrophy [37, 38], initiation of cardiac myocyte DNA synthesis [38]and NO production [39]. Other specific alterations in gene expression have been noted, including activation of atrial natriuretic peptide (ANP) gene and depression of genes encoding sarcoplasmic calcium ATPase, calcium release channel, voltage-dependent calcium channel and phospholamban [37, 40, 41]. It is not currently known if these alterations in gene expression are mediated via an NF-B dependent pathway. In the case of cytokine-induced NO production, this has been linked directly to the activation of the inducible nitric oxide synthase (iNOS) gene [42]by NF-B, as blocking NF-B activity using pyrrolidine dithiocarbamate (PDTC) eliminates iNOS gene activation [5, 42, 43], in agreement with studies in other cells where NF-B binding was shown to be required for activation of the iNOS gene promoter [13]. Further studies are now required to determine if similar direct mechanisms operate on the other genes influenced by IL-1β-stimulated myocytes.

The depletion of IB- in cardiac myocytes as demonstrated here follows the rapid, transitory response noted for most cell types [33, 44, 45]. Furthermore, the accumulation of NF-B DNA-binding activity in the nucleus appears to mirror this depletion, suggesting that IB- may be the principal regulator of NF-B activity, at least in response to IL-1β. The precise role of IB-β remains to be determined. However, a recent study has identified a number of potentially important functional differences between IB- and IB-β [46]. In particular, IB- is probably a stronger inhibitor of NF-B and can act to remove bound NF-B from the nucleus, whereas IB-β cannot. IB-β activity may also be influenced by phosphorylation and interaction with other nuclear factors, including HMGI. Our data show that IB- and IB-β respond differently to IL-1β stimulation in cardiac myocytes. This is in line with previous studies in lymphocytes, which demonstrated a slower, more persistent depletion of IB-β in response to a variety of stimuli including IL-1β [23], suggesting that it is involved in more long-term NF-B activation. The precise signal transduction pathways that result in IB activation remain to be determined but are probably different for IB- and IB-β. For example, while tumour necrosis factor- (TNF-) elicits a strong depletion of IB- in both endothelial and Jurkat cells, IB-β is only responsive in Jurkat cells [23, 47]. In our experiments, IL-1β resulted in only a partial depletion of IB-β in cardiac myocytes. Further experiments are now required to determine the functional significance of IB-β in NF-B regulation in these cells.

Recent studies have shown that inhibiting NF-B activity through the use of a decoy oligonucleotide results in a significant reduction in myocardial damage following coronary artery ligation [4]. The mechanisms involved in this are currently unknown but may involve direct inhibition of NF-B in cardiac myocytes. In this context, it is of interest to note that NF-B activation has been demonstrated in isolated cardiac myocytes in response to hydrogen peroxide [7]. Moreover, as it has been shown that hypoxia alone does not activate NF-B [6], suggesting that reperfusion injury and not ischemia is the primary trigger for activation. Given the potential therapeutic value of these observations, detailed experiments are now required to examine the precise signal transduction pathways involved as well as the downstream targets of NF-B.

Time for primary review 32 days

	Acknowledgements

 
We are grateful to Dr. Nandor Marczin for his inspiration and enthusiasm, to Dr. Paul Burton for assistance in preparing protein extracts, to Kim Dellow for assistance with EMSA and to Dr. Leslie Wong (MRC Fetal Tissue Bank) for assistance with human fetal heart samples. This work was supported by the British Heart Foundation (FS297).

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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