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Cardiovascular Research 2004 64(2):289-297; doi:10.1016/j.cardiores.2004.06.029
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Copyright © 2004, European Society of Cardiology

Attenuation of NF-{kappa}B signalling in rat cardiomyocytes at birth restricts the induction of inflammatory genes

Nora Goren, Jimena Cuenca, Paloma Martín-Sanz and Lisardo Boscá*

Instituto de Bioquímica, Centro Mixto CSIC-UCM, Facultad de Farmacia, Universidad Complutense, Madrid 28040, Spain
Centro Nacional de Investigaciones Cardiovasculares, Ronda de Poniente 5, Tres Cantos, Madrid 28760, Spain

* Corresponding author. Instituto de Bioquímica, Centro Mixto CSIC-UCM, Facultad de Farmacia, Universidad Complutense, Madrid 28040, Spain. Tel.: +34 91 3941853; fax: +34 91 3941782. Email address: lbosca{at}cnic.es, boscal{at}farm.ucm.es

Received 24 March 2004; revised 10 June 2004; accepted 30 June 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 
Objectives and background: The expression of inflammatory genes in the heart plays an important role in organ dysfunction. However, the contribution of cardiomyocytes to this process, and in particular to the synthesis of high concentrations of nitric oxide and prostaglandins, has not been analyzed in detail. For this reason, cultured isolated cardiomyocytes were used to evaluate the response to pro-inflammatory stimuli.

Methods and results: Isolated cultured foetal, neonatal, and adult rat cardiomyocytes were stimulated with lipopolysaccharide and cytokines, and the expression of nitric oxide synthase-2 (NOS-2) and cyclooxygenase-2 (COX-2) in these cells was investigated. Only cultured foetal cardiomyocytes expressed NOS-2 and COX-2 under these conditions, whereas the neonatal counterparts required various days in culture to gain this response. Analysis of the NF-{kappa}B signalling pathway showed an impaired activation of I{kappa}B kinase in response to lipopolysaccharide and cytokines in cells maintained in culture for 1 day. These data were confirmed by DNA microarray analysis. However, other early signalling pathways, such as the p38 and Erk MAPKs, were not affected by the time in culture.

Conclusions: Neonatal and adult cardiomyocytes are resistant to the expression of pro-inflammatory genes due to impairment in the activation of I{kappa}B kinase, a process that might contribute to preventing rapid organ dysfunction in the course of various inflammatory pathologies, such as septic shock and myocarditis.

KEYWORDS Inflammation; Myocytes; Nitric oxide; Prostaglandins; Oxygen radicals

Abbreviations: COX, cyclooxygenase • CK, cytokines TNF-{alpha} and IL-1β • IL-1, interleukin 1 • IKK, I{kappa}B kinase • LPS, lipopolysaccharide • NF-{kappa}B, nuclear factor {kappa}B • NOS, nitric oxide synthase • PG, prostaglandin • TNF-{alpha}, tumour necrosis factor {alpha}


   1. Introduction
Top
 Abstract
 1. Introduction
2. Material and methods
 3. Results
 4. Discussion
 References
 
In the course of systemic bacterial infection a cascade of inflammatory responses occurs. This involves the recognition of pathogen associated motifs and the release of pro-inflammatory cytokines and chemokines [1–4]. In heart, this situation is even more complex due to the infiltration of inflammatory cells to the organ; this infiltration making the study of the expression of genes mediating inflammation in cardiomyocytic cells difficult. Several groups have described the presence of NOS-2 in interstitial or infiltrating cells [4–6], and this induction of NOS-2 plays an important role in the pathogenesis of cardiac dysfunctions, such as myocarditis, septic shock and myocardial infarction [6]. Cultured cardiac myocytes constitute an appropriate ex vivo model for studying the specific response of cardiac cells to pro-inflammatory and pathogenic stimuli. The ability of cardiomyocytes to respond to cytokines and bacterial molecules has been reported by several authors [2,7,8]. However, in isolated cardiac cells from neonatal or adult animals there are discrepancies regarding the conditions required to observe the expression of genes involved in the inflammatory response, such as NOS-2, COX-2 and matrix metalloproteinases [4,9,10].

Activation of NF-{kappa}B in cardiomyocytes has been shown to be an important mediator of the inflammatory response in the heart and of organ damage, and the presence of p65, p50 and I{kappa}B{alpha} and I{kappa}Bβ in the myocardium has been described through development, with a decrease from the 10 day post-natal to the adult [11,12]. For example, when oligonucleotide sequences corresponding to the {kappa}B motif were administered in an animal model of autoimmune myocarditis, this reduced the expression of NF-{kappa}B-regulated genes including NOS-2, pro-inflammatory cytokines (TNF-{alpha} and IL-1β) and adhesion molecules, protecting against heart dysfunction [13]. Similar results have been obtained after modulation of NF-{kappa}B activity with other strategies [14–16]. It is clear therefore that the NF-{kappa}B pathway activates molecules that regulate the onset of the inflammatory response in heart. We have investigated the ability of cultured cardiomyocytes from foetal, neonatal, and adult rats to respond to proinflammatory stimuli and to express enzymes involved in the release of inflammatory mediators. Under these conditions the contribution of inflammatory cells is avoided. Our data show that foetal cardiomyocytes readily express NOS-2 and COX-2 after LPS and pro-inflammatory challenge. In contrast, neonatal and adult cardiomyocytes are unable to express these enzymes except after a prolonged period of cell culture. This comparatively weak response appears to be due to the lower activation of the I{kappa}B kinase/NF-{kappa}B pathway observed in neonatal and adult cardiomyocytes, which might protect the organ against the deleterious effects of rapid expression of inflammatory genes.


   2. Material and methods
Top
 Abstract
 1. Introduction
 2. Material and methods
3. Results
 4. Discussion
 References
 
2.1 Chemicals
LPS from Salmonella typhimurium and all other reagents not specified were from Sigma (St. Louis, MO). Cytokines were from Roche (Mannheim, Germany). Collagenase type II was from Worthington (Lakewood, NJ). Antibodies were obtained from Santa Cruz Laboratories (Santa Cruz, CA) or Calbiochem (San Jose, CA). Tissue culture dishes were from Falcon (Lincoln Park, NJ) and culture media were from BioWhittaker (Walkersville, MD).

2.2 Isolation and culture of rat foetal, neonatal, and adult cardiomyocytes
The investigation conforms to the Guide for the Care and Use of Laboratory Animals (NIH Publ. 85-23, revised 1996). Cardiomyocytes from the hearts of 20-, 21-, and 22-day-old foetuses were isolated after removal of foetuses from pregnant Albino Wistar rats (300–350 g). Neonatal cardiomyocytes were prepared from the hearts of 1- and 5-day-old animals. In both these cases the tissues were chopped, disaggregated mechanically, and digested for 15 min at 37 °C with 0.8% type II collagenase in PBS, according to a previous protocol of successive digestions [17]. The cell pellets were resuspended and maintained in 250 ml flasks containing 50 ml of DMEM-F-12:M-199 medium (4:1, vol/vol), supplemented with 10% FCS and antibiotics. After incubation for one hour, the cell suspension was sedimented by centrifugation at low speed (80 g), and cells were distributed in plates pre-coated with a 2% solution of gelatine. After overnight incubation to favour cardiomyocyte adhesion, the dishes were washed with PBS and the medium was replaced. Usually, cells were plated in 6 cm culture dishes (1–2x106 cells) or 24 multiwell tissue culture plates (2x105 cells). When adult cardiomyocytes were prepared, the heart was perfused as described [17,18], and isolated cells were treated as described for the foetal and neonatal cardiomyocytes. Cells were treated for the indicated periods of time with LPS, CK (IL-1β and TNF-{alpha}) and IFN-{gamma}.

2.3 Preparation of cytosolic and nuclear extracts
Cultured cells were washed with PBS, scraped off the dishes in ice-cold PBS, and centrifuged. Cell pellets were homogenized in 200 µl of buffer A (10 mmol/l HEPES; pH 7.9, 1 mmol/l EDTA, 1 mmol/l EGTA, 10 mmol/l KCl, 1 mmol/l DTT, 0.5 mmol/l phenylmethyl sulfonyl fluoride, 40 µg/ml leupeptin, 2 µg/ml tosyl-lysyl-chloromethane, 5 mmol/l NaF, 1 mmol/l NaVO4, 10 mmol/l Na2MoO4), and NP-40 was added to reach 0.5% (vol/vol). After 15 min at 4 °C, the tubes were gently vortexed for 15 s, and nuclei were collected by centrifugation at 8000xg for 15 min. The supernatants were stored at –80 °C (cytosolic extracts) and the pellets were resuspended in 50 µl buffer A supplemented with 20% (vol/vol) glycerol and 0.4 M KCl, and mixed for 30 min at 4 °C. Nuclear proteins were obtained by centrifugation at 13,000xg for 15 min, and aliquots of the supernatant (nuclear extracts) were stored at –80 °C. For Western blot analysis, cytosolic and nuclear proteins were boiled in Laemmli sample buffer, and equal amounts of protein (30–15 µg) were separated by 10% SDS-PAGE [19].

2.4 Electrophoretic mobility shift assays (EMSAs)
The oligonucleotide sequence corresponding to the NF-{kappa}B site of the rat COX-2 promoter (5'-GGCAAGGGGATTCCCTTAGTT-3') [20] was annealed with the complementary sequence by incubation for 5 min at 85 °C in 10 mmol/l Tris–HCl; pH 8.0, 50 mmol/l NaCl, 10 mmol/l MgCl2, 1 mmol/l DTT. Aliquots (100 ng) were end-labelled with Klenow enzyme fragment in the presence of 50 µCi of [{alpha}-32P]dCTP and the other unlabeled dNTPs in a final volume of 50 µl. 5x104 dpm of the DNA probe was used for each binding assay: 5 µg of nuclear protein were incubated for 15 min at 4 °C with the probe and with 1 µg of poly (dI-dC), 5% glycerol, 1 mmol/l EDTA, 10 mmol/l KCl, 5 mmol/l MgCl2, 1 mmol/l DTT, and 10 mmol/l Tris–HCl (pH 7.8) in a final volume of 20 µl. The DNA–protein complexes were separated on native 6% polyacrylamide gels in 0.5% Tris–borate–EDTA buffer [21]. Supershift assays were performed by incubating the nuclear extracts (3 µg protein) with the indicated antibodies against NF-{kappa}B proteins (p50 and p65) prior to the addition of the probe.

2.5 Western blot analysis
The protein levels of COX-2, NOS-2, I{kappa}B{alpha} (and phospho-ser32-I{kappa}B{alpha}), I{kappa}Bβ, IKK, Erk (and phospho-Erk), p38 (and phospho-p38), p65, Sp1, and {alpha}-actin were determined in cytosolic or nuclear extracts as indicated. Equal amounts of protein (15–30 µg) were size-fractionated in a 10% acrylamide gel, transferred to a Hybond P membrane (Amersham), and, after blocking with 5% non-fat dry milk, were incubated with the corresponding antibodies and visualized by ECL. Different exposure times were performed with each blot to ensure the linearity of the band intensities. Band intensities were measured on a densitometric scanner (Amersham), and expressed in arbitrary units.

2.6 Determination of cytokines and metabolites
PGE2 levels in culture media were determined by a specific enzyme immunoassay in accordance with the manufacturer's instructions (Biotrak, Amersham). To determine the amount of NO released into the medium, nitrate was reduced to nitrite, and this was measured spectrophotometrically by the Griess reaction, as described [22]. Real-time measurement of NO synthesis was performed cells loaded with DAF-FM, and following the controls and recommendations of the supplier (Molecular Probes, Eugene, OR).

2.7 Measurement of IKK activity
Cardiomyocytes (5x106) were homogenized in 1 ml of buffer A and centrifuged for 10 min in a microcentrifuge. IKK was immunoprecipitated from the supernatant with 1 µg of a specific Ab. After washing with 4 ml of buffer A, the pellet was resuspended in kinase buffer (20 mmol/l Hepes, pH 7.4; 0.1 mmol/l EDTA, 100 mmol/l NaCl, 1 mmol/l DTT, 0.5 mmol/l PMSF, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml TLCK, 5 mmol/l NaF, 1 mmol/l NaVO4, 10 mmol/l Na2MoO4, and 10 nmol/l okadaic acid). Kinase activity was assayed in 100 µl kinase buffer containing 100 ng precipitated protein. In assays where myelin basic protein (MBP) was used as substrate, the reaction contained 50 µM [{gamma}-32P]ATP (0.5 µCi). When the substrate was I{kappa}B{alpha}, 1 mmol/l MgATP was used. Aliquots of reactions were stopped with 1 ml of ice-cold buffer A supplemented with 5 mmol/l EDTA, and analyzed by SDS-PAGE and autoradiography or by Western blot using anti-P-S32-I{kappa}B{alpha} Ab.

2.8 DNA microarray analysis
A pool of three independent cardiomyocyte preparations from 5-day-old rats that had been maintained in culture for 1 or 5 days were stimulated for 2 h with LPS and CK. RNA was then extracted and analyzed in a 2100 Bioanalyzer following the instructions of the manufacturer of the array (Affymetrix). Fragmentation of biotin-labelled complementary RNA (cRNA) was performed at 94 °C using 40 mmol/l Tris–acetate, pH 8.1; 100 mmol/l KOAc, and 30 mmol/l MgOAc. Samples were hybridized to Affymetrix GeneChip Rat expression probe array 230A. The data were analyzed with the GeneChip Analysis Suite software.

2.9 Confocal microscopy
Cardiomyocytes were grown on gelatine-coated cover slips and incubated for the indicated times with the indicated stimuli. After washing the cover slips twice with ice-cold PBS, the cells were fixed (4% paraformaldehyde in PBS, 15 min; methanol at –20 °C, 15 min), blocked for 30 min at room temperature with 3% BSA, and incubated for 60 min with the primary Ab (usually at 1:200–400 dilution). After washing with PBS, the fixed cells were incubated with anti-rabbit IgG Ab (1:500) conjugated to Cy3 (Amersham). The cells were visualized on an MRC-1024 confocal microscope (Bio-Rad), and the fluorescence was measured using LaserSharp software (Bio-Rad).

2.10 Transient transfection of cardiomyocytes
Primary cultures of neonatal cardiomyocytes were transfected for 6 h with 5 µl of JetPEI and 1 µg of DNA per dish (2.5 cm) in the presence of 1% FCS, in accordance with the supplier's instructions (PolyTransfection, Illkirch, France). After transfection, cells were incubated overnight in 2 ml of culture medium plus 2% FCS. The plasmids used for transfection were: ({kappa}B)3ConA-LUC, which contains three copies of the {kappa}B motif from the HIV LTR enhancer linked to the minimal conalbumin A promoter; pNOS-2-LUC, containing a 1 kb fragment of the 5'-flanking region of the NOS-2 gene and two {kappa}B motifs fused to a LUC reporter; pCOX-2-LUC, containing a 0.5 kb fragment of the COX-2 promoter (nucleotides –446 to+32) fused to a LUC reporter [22]. The promoterless pLUC plasmid was used as a control, and was not unaffected by experiments. Exogenous activation of NF-{kappa}B signalling was achieved by co-transfecting the cells with 0.5 µg per plate of an expression vector for p65 (pCMV-p65). Co-transfection with a β-galactosidase reporter vector under the control of the cytomegalovirus promoter (pCMV-β-gal; Clontech, Palo Alto, CA), was used to monitor the efficiency of transfection. Luciferase and β-galactosidase activities were measured by use of a commercial kit (Promega). Plasmids were purified on EndoFree Qiagen columns (Hilden, Germany) [19,21].

2.11 Data analysis
Data were analyzed with SPSS software (Chicago). ANOVA was used to evaluate statistical significance for the indicated experiments. Results are expressed as the mean±SE of the indicated number of experiments. Statistical significance was estimated with Student's t-test for unpaired observations. A P value of less than 0.05 was considered significant. For the analysis of the Western blots, we used linear correlations between increasing amounts of protein and signal intensity.


   3. Results
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
4. Discussion
 References
 
3.1 Time-dependent expression of inflammatory genes by isolated neonatal cardiomyocytes
Cardiomyocytes were prepared from neonatal animals (5 days old), and were maintained in culture for up to 8 days with no significant loss in the percentage of cells positive for {alpha}-actin; a marker of this cell type. At different days of culture, cells were stimulated for 24 h with LPS and pro-inflammatory cytokines. As Fig. 1A shows, the capacity of cells to express COX-2 and NOS-2 was observed only after at least 3 days in culture. When cardiomyocytes maintained in culture for 5 days were treated with combinations of different pro-inflammatory stimuli, cooperation was observed in the induction of COX-2 and NOS-2 expression, exhibiting a high sensitivity to IL-1β challenge (Fig. 1B). Consistent with these data, detection of NO synthesis with the NO-sensitive probe DAF-FM showed a clear fluorescence in cells after 3 days in culture, with the majority of cells responding by day 4, but not at earlier times (Fig. 1C). Fig. 1D shows the accumulation of nitrite plus nitrate (NOx) and prostaglandin E2 (PGE2) in the culture media of cells stimulated after 1 or 5 days in culture. A clear response was observed only in cells cultured for 5 days prior to stimulation.


Figure 1
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  		Fig. 1 Delayed expression of COX-2 and NOS-2 in neonatal cardiomyocytes. Cells were prepared from hearts of 5-day old rats, and maintained in culture for the indicated periods of time. Expression of NOS-2 and COX-2 at different days of culture was determined by Western blot, after 24 h of stimulation with LPS (1 µg/ml) and CK (TNF-{alpha} and IL-1β, each at 10 ng/ml) (panel A). The effect of pro-inflammatory stimuli on the expression of these genes was determined in cardiomyocytes maintained for 5 days in culture and then stimulated for 24 h with these factors. IFN{gamma} was used at 20 units/ml (panel B). After labelling the cells with the NO-sensitive probe DAF-AM, the fluorescence was visualized by confocal microscopy (panel C). The synthesis and release of NO and PGs into the medium by cardiomyocytes maintained in culture for 1 or 5 days was determined by the measurement of nitrite plus nitrate, and PGE2, respectively (panel D). Results show the mean±SE of four experiments (a.u., arbitrary units). *p<0.001 vs. the corresponding non-stimulated cells, or at day 1.

 
3.2 Foetal cardiomyocytes express NOS-2 and COX-2 upon pro-inflammatory challenge
In contrast to their neonatal counterparts, cardiomyocytes prepared from 21-day foetuses and maintained in culture for 24 h responded rapidly to LPS and CK, with a marked induction of NOS-2 and COX-2 (Fig. 2A). The expression of NOS-2 and COX-2 was very NF-{kappa}B-dependent, and a rapid and almost complete degradation of I{kappa}B{alpha} was observed 60 min after challenge. The levels of I{kappa}Bβ also decreased after pro-inflammatory stimulation, although the recovery was delayed with respect to I{kappa}B{alpha}. The expression of COX-2 and NOS-2 was confirmed by immunocytochemistry, and as Fig. 2B shows, more than 80% of cells were positive for the expression of these proteins after stimulation with LPS and CK. In addition to this, the accumulation of NOx and PGE2 in the culture media confirmed that both NOS-2 and COX-2 were catalytically active, and was in the range measured in Fig. 1D for 5-day-old neonatal cardiomyocytes (not shown). Moreover, when the responses of foetal, neonatal, and adult cardiomyocytes to pro-inflammatory stimuli were analysed at 24 h after preparation, we found that NOS-2 was expressed in cells from 20-, 21-, and 22-day-old foetuses, but declined rapidly by 1 day after birth, and was undetectable in cells obtained after 5 or 60 days (Fig. 2C).


Figure 2
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  		Fig. 2 Foetal cardiomyocytes express NOS-2 and COX-2 after pro-inflammatory stimulation. Foetal cardiomyocytes were prepared from 21-day old rat foetus hearts, and after overnight incubation to favour adhesion the cells were challenged for 24 h with TNF-{alpha} (10 ng/ml), IL-1β (10 ng/ml), LPS (1 µg/ml), IFN-{gamma} (20 units/ml) or combinations of these. The expression levels of NOS-2, COX-2 and {alpha}-actin were determined by Western blot. The kinetics of I{kappa}B{alpha} and I{kappa}Bβ degradation and resynthesis were analyzed (panel A). The percentage of cells expressing NOS-2, COX-2 and {alpha}-actin was determined by immunocytochemistry (n=80), and a representative staining is shown (panel B). Cardiomyocytes prepared from foetal (20, 21, and 22 days gestation) and post-birth (1, 5 and 60 days) rats were maintained in culture for 24 h and stimulated with LPS, CK (i.e. IL-1β and TNF-{alpha}, at 10 ng/ml) and IFN-{gamma} for 24 h. The expression of NOS-2 was determined by Western blot. Results show the mean±SE of three experiments. *p<0.05, **p<0.001 vs. the corresponding non-stimulated cells; ap<0.05 vs. the CK condition (panel B).

 
3.3 NF-{kappa}B activity is reduced in neonatal cardiomyocytes
To explore potential mechanisms that might underlie the failure of recently isolated postnatal cells to express NOS-2 and COX-2, we investigated signalling through the NF-{kappa}B pathway. Fig. 3 (A–B) shows that the activation of IKK by LPS and cytokines was much weaker in cells after 1 day in culture than after 5 days; revealed by the low capacity of the immunoprecipitated enzyme to phosphorylate MBP, or I{kappa}B{alpha} at serine 32. In agreement with these data, the binding of NF-{kappa}B proteins to a consensus DNA sequence, the degradation of I{kappa}B{alpha} and I{kappa}Bβ, and the translocation of p65 to the nucleus were all notably lower in cells cultured for only 1 day (Fig. 3C). Pharmacological inhibition of NF-{kappa}B activity with the translocation inhibitor SN50 or the proteasome inhibitors lactacystine or MG132 prevented synthesis of NO and PGE2, confirming the dependence of NOS-2 and COX-2 expression on NF-{kappa}B (Fig. 3D). To investigate whether the weakened NF-{kappa}B signalling was specific or symptomatic of a more general dampening of stress-response pathways, we examined p38 and Erk MAPKs activation after LPS and CK challenge. As Fig. 3E shows the activation of these pathways did not differ between cells maintained in culture for 1 or 5 days after preparation, suggesting a specific action of the NF-{kappa}B pathway in the attenuation of NOS-2 and COX-2 expression.


Figure 3
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  		Fig. 3 The NF-{kappa}B pathway is attenuated in recently isolated neonatal cardiomyocytes. Cardiomyocytes were prepared from hearts of 5-day-old rats. Cells cultured for 1 or 5 days were stimulated with 1 µg/ml of LPS and CK, and at the indicated times cytosolic and nuclear extracts were prepared. The activity of IKK was measured after immunoprecipitation with specific antibodies, using MBP ([32P]-incorporation) and I{kappa}B{alpha} (P-S32-I{kappa}B{alpha} detection by Western blot) as substrates (panel A). The densitometric analysis (mean of three experiments) is shown (panel B). The NF-{kappa}B activity was determined by EMSA, and the levels of p65 and Sp1 (control of lane loading) in the nuclear protein fraction, and I{kappa}B{alpha}, I{kappa}Bβ and {alpha}-actin in the cytosol, were determined by Western blot (panel C). The effect of the NF-{kappa}B inhibitor SN50 (25 µM) and the proteasome inhibitors lactacystine (20 µM) and MG132 (20 µM) on NO and PGE2 synthesis (24 h accumulation) were determined (panel D). The activation of Erk and p38 MAPKs was determined by Western blot (panel E). Results show a representative experiment of three performed (A, C, E), and the means±SE of three experiments (B,D). *p<0.001 vs. the corresponding control cells.

 
To gain further insight into this differential activity of the NF-{kappa}B pathway in neonatal cardiomyocytes with the time in culture, we used an Affymetrix microarray (rat expression 230A) to compare the profile of expression after stimulation with LPS and CK for 2 h. Fig. 4 shows data for a selection of genes whose expression was altered more than 2-fold at 1 or 5 days in culture. A cluster of genes involved in NF-{kappa}B signalling, genes for pro-inflammatory cytokines, chemokines and their receptors, and genes for enzymes involved in the synthesis of inflammatory mediators (NOS-2, COX-2) or extracellular matrix remodelling were all increased in cells activated after 5 days in culture. These changes were either much smaller or below the threshold at day 1 in culture. Particularly, the levels of the p50 and p65 precursors p105 and p100, as well as I{kappa}Bβ and A20 increased notably after stimulation of cells maintained 5 days in culture. Also, the DNA array data show an increase in the expression levels of pro-inflammatory cytokines and some chemokines (LIX), as well as in the expression of receptors for normal and oxidized LDL, TNF-{alpha}, IL-1β and PDF.


Figure 4
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  		Fig. 4 Transcriptional profiling of cultured neonatal cardiomyocytes. Cells were cultured for 1 or 5 days, and stimulated for 2 h in the presence or absence of LPS and CK. A selection of genes regulated by NF-{kappa}B and involved in inflammation is shown. Only genes whose transcription increased/decreased after activation two-fold in one condition are shown.

 
These results prompted us to evaluate the activity of a {kappa}B responsive sequence and of the promoter regions of the NOS-2 and COX-2 genes in luciferase-reporter transfection experiments in cardiomyocytes. These experiments were conducted in the absence or presence of a co-transfected p65 expression vector (see Materials and methods). As Fig. 5 shows, upon activation, neonatal cardiomyocytes maintained in culture for 1 day exhibited about one third of the luciferase activity of cells maintained in culture for 5 days. The contrast was greater in the cases of the NOS-2 and COX-2 promoters, where the activity was negligible at 1 day. Cotransfection of a p65 expression vector conferred responsiveness to cells at one day in culture. This strongly suggests that the NF-{kappa}B pathway is insufficiently activated in recently isolated neonatal cardiomyocytes to promote NOS-2 and COX-2 expression.


Figure 5
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  		Fig. 5 Regulation of the {kappa}B, and NOS-2 and COX-2 promoter activity in cardiomyocytes. Neonatal cells maintained in culture for 1 or 5 days were transfected with an NF-{kappa}B-responsive construct or the NOS-2 or COX-2 promoters, linked to a luciferase reporter gene, with or without a p65 expression vector. Ectopic expression of β-galactosidase was used to normalize the efficiency of transfection. Cells were stimulated for 18 h with LPS and CK, and the luciferase and β-galactosidase activities were measured. Results show the mean±SE of three experiments. *p<0.05, **p<0.01 vs. the corresponding control condition; ap<0.01 vs. the corresponding control condition in cells at day 1 of culture.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
References
 
In this work we have analysed in some detail the sequence of events that occur in isolated cardiomyocytes after LPS and CK administration. Our data show that cardiomyocytes exhibit very specific responses according to the age of the animal, with foetal cells being more responsive than their neonatal and adult counterparts, specifically in terms of the expression of NF-{kappa}B-dependent genes, for example, NOS-2 and COX-2. Moreover, NOS-2 expression is detected in the heart after myocardial infarction, and the associated NO synthesis seems to correlate with contractile dysfunction, local inflammation, and promotion of cardiomyocyte apoptosis and necrosis [23,24]. However, studies from our group indicate that early NOS-2 expression occurs mainly in infiltrating cells, not in cardiomyocytes. In fact, in hearts from control mice we have observed the presence of infiltrated cells that are MHC class II positive, but negative for NOS-2 or COX-2 expression (work in progress).

Previous work reported the expression of NOS-2 in cardiomyocytes from 1-day-old animals challenged with high concentrations of LPS (in the 1–10 µg/ml range), and this process exhibited an important synergism with IFN-{gamma} incubation [9]. This was not the case in our experiments with cells from 5-day-old animals that required several days in culture to gain the ability to express important inflammatory genes. Furthermore, in addition to the initial attenuated response, the most potent co-inducer together with LPS in our experiments was IL-1β not IFN-{gamma}. Supporting this observation, there are some indications of specific responses to pro-inflammatory insults in the heart depending on the age of the animals [17].

Interestingly, all cultured cardiomyocytes, including adult cells, and irrespective of the period in culture, responded rapidly to LPS by activating the well known p38 and Erk MAP kinase pathways, but exhibited differences in the activation of the NF-{kappa}B pathway. The role of NF-{kappa}B as an integrator of host defence, inflammatory and oxidative stress signalling has been also observed in in vivo experiments in which animals were infused with decoy oligonucleotides against NF-{kappa}B [13]. This provoked a 30–50% reduction in the expression of NOS-2, IL-2, TNF-{alpha}, and adhesion molecules such as ICAM-I. Our data from the microchip DNA array show that the transcriptional activity of NF-{kappa}B-dependent genes is significantly weaker in cardiomyocytes from neonates after 1 day in culture than at day 5 of plating. In agreement to these data a much lower activation of IKK and targeting of I{kappa}B{alpha} and I{kappa}Bβ. This reduced IKK activation cannot be attributed to differences in the amount of kinase, because the levels determined by Western blot were similar. One possible beneficial consequence of reduced IKK activation in cardiomyocytes might be to prevent the establishment of an initial pro-inflammatory situation as it has been reported in several pathophysiological models of heart dysfunction, such as unstable angina, where NF-{kappa}B and also AP-1 are rapidly activated favouring a pro-inflammatory situation [25]. Indeed, it has been suggested that excessive delivery of NO by inflammatory cells results in profound cellular disturbances, leading to heart failure [26] and, therefore, a moderate expression (via the NF-{kappa}B-dependent pathway) might even exert beneficial effects [26,27]. Regarding the expression of COX-2 after heart injury, some confusion exists about the beneficial or detrimental effects of the expression and/or inhibition with selective drugs. In the case of ischemia/reperfusion injury, the expression of COX-2 exerts cardioprotective roles [28], whereas after myocardial infarction, a condition that triggers COX-2 expression, inhibition of its activity improves cardiac function [29–31].

In summary, our data demonstrate a specific protection of non-foetal cardiomyocytes against the expression of genes involved in the inflammatory response. Assessment of the specific mechanisms that contribute to this protection in healthy animals might help in the development of strategies to decrease heart injury in many different pathophysiological situations such as myocarditis, myocardial infarction and idiophatic cardiomyopathy.


   Acknowledgements
 
The authors thank Dr Simon Bartlett for help in the preparation of the manuscript; this work was supported by grants SAF2002-00783 and 08.3/0008/2001 from CICYT and Comunidad de Madrid, respectively.


   Notes
 
Time for primary review 20 days.


   References
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 

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