Skip Navigation

Cardiovascular Research 2005 66(3):520-529; doi:10.1016/j.cardiores.2005.02.007
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Kawamura, N.
Right arrow Articles by Sunagawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kawamura, N.
Right arrow Articles by Sunagawa, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2005, European Society of Cardiology

Blockade of NF-{kappa}B improves cardiac function and survival without affecting inflammation in TNF-{alpha}-induced cardiomyopathy

Natsumi Kawamuraa, Toru Kubotaa,*, Shunichi Kawanoa, Yoshiya Mondena, Arthur M. Feldmanb, Hiroyuki Tsutsuia, Akira Takeshitaa and Kenji Sunagawaa

aDepartment of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
bDepartment of Medicine, Jefferson Medical College, Philadelphia, PA, USA

* Corresponding author. Tel.: +81 92 642 5360; fax: +81 92 642 5374. Email address: kubotat{at}cardiol.med.kyushu-u.ac.jp

Received 18 October 2004; revised 7 February 2005; accepted 7 February 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: NF-{kappa}B, a key transcription factor that regulates inflammatory processes, has been shown to be activated in the failing human heart with enhanced expression of proinflammatory cytokines. In the present study, we assessed the hypothesis that cardiotoxic effects of proinflammatory cytokines are mediated by the activation of NF-{kappa}B.

Methods: Transgenic mice with cardiac-specific overexpression of TNF-{alpha} were used as a model of cytokine-induced cardiomyopathy. To block the activation of NF-{kappa}B, transgenic mice (TG/p50+/+) were crossed with knockout mice in which the p50 subunit of NF-{kappa}B was disrupted (WT/p50–/–).

Results: The electrophoretic mobility shift assay demonstrated that NF-{kappa}B was activated in the myocardium of TG/p50+/+ mice, while it was completely abolished in TG/p50–/– mice. Male TG mice died of congestive heart failure earlier than females, where the disruption of the p50 subunit significantly improved the survival. Compared with TG/p50+/+ mice, TG/p50–/– mice showed a significant reduction of ventricular dilatation and hypertrophy with preserved fractional shortening. Although the myocardial expression of proinflammatory cytokines or infiltration of inflammatory cells was not affected, increased expression and activity of MMP-9 were significantly suppressed in TG/p50–/– mice.

Conclusion: Blockade of NF-{kappa}B activation did not ameliorate myocardial inflammation but improved cardiac function and survival in male TNF-{alpha} TG mice. An inhibition of NF-{kappa}B may be a new therapeutic strategy for cardiac remodeling and heart failure, especially when proinflammatory cytokines are activated.

KEYWORDS Nuclear factor-{kappa}B; Cytokines; Tumor necrosis factor-{alpha}; Matrix metalloproteinases; Transgenic animal models; Myocarditis; Heart failure; Cardiac remodeling


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Tumor necrosis factor (TNF)-{alpha} is a proinflammatory cytokine, which exerts a wide range of biological activities [1]. TNF-{alpha} may play an important role in the pathogenesis of congestive heart failure for the following reasons. First, plasma levels of TNF-{alpha} are elevated in patients with congestive heart failure [2,3]. Second, the failing human heart expresses a substantial amount of TNF-{alpha} [4,5]. Third, studies in vitro have shown that TNF-{alpha} suppresses cardiac contractility [6,7], provokes myocardial hypertrophy [8,9], and induces apoptosis in cardiac myocytes [10]. To investigate the pathophysiological significance of myocardial production of TNF-{alpha} in vivo, we have made transgenic mice that overexpress TNF-{alpha} specifically in the heart under the control of {alpha}-myosin heavy chain promoter [11]. These mice present myocardial inflammation, ventricular dilatation, and congestive heart failure. Furthermore, male mice die younger than females [12,13]. Treatment with soluble TNF receptors reverse myocardial inflammation, extracellular matrix remodeling, and ventricular dysfunction in these mice [14,15]. Several aspects of these results have since been confirmed by another laboratory [16]. Thus, myocardial production of TNF-{alpha} may play an important role in the development of congestive heart failure. However, the mechanisms by which TNF-{alpha} damages the myocardium remain undetermined.

Nuclear factor-kappa B (NF-{kappa}B) is a key transcription factor that regulates inflammatory processes [17]. Many stimuli activate NF-{kappa}B, including proinflammatory cytokines, lipopolysaccharide, and reactive oxygen species. Activation of NF-{kappa}B involves the phosphorylation and subsequent proteolytic degradation of the inhibitory protein I{kappa}B by specific I{kappa}B kinases. The free NF-{kappa}B (typically, a heterodimer of p50 and p65) then passes into the nucleus, where it binds to {kappa}B sites in the promoter regions of genes for inflammatory proteins such as TNF-{alpha}, inducible nitric oxide synthase, and adhesion molecules. Thus the activation of NF-{kappa}B leads to a coordinated increase in the expression of many genes whose products mediate inflammatory and immune responses [17]. Furthermore, the activation of NF-{kappa}B may also play an important role in the pathogenesis of cardiac remodeling and heart failure [18], since recent in vitro studies have demonstrated that activation of NF-{kappa}B is required for hypertrophic growth of primary rat neonatal ventricular cardiomyocytes in response to angiotensin II, phenylephrine, and endothelin-1 [19,20]. Therefore, blockade of NF-{kappa}B may be a new therapeutic strategy for heart failure by attenuating myocardial inflammation and hypertrophy.

Based on these backgrounds, the present study was designed to assess the hypothesis that cardiotoxic effects of proinflammatory cytokines are mediated by the activation of NF-{kappa}B. To block the activation of NF-{kappa}B, transgenic mice with cardiac-specific overexpression of TNF-{alpha} [11] were crossed with knockout mice in which the p50 subunit of NF-{kappa}B was disrupted (WT/p50–/–) [21]. Although the blockade of NF-{kappa}B did not ameliorate myocardial inflammation, it significantly improved cardiac function and survival in male TNF-{alpha} transgenic mice. These results support the hypothesis that the activation of NF-{kappa}B may play an important role in myocardial dysfunction and remodeling besides promoting inflammation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animal model
Transgenic mice with cardiac-specific overexpression of TNF-{alpha} (TNF TG) [11] and wild-type littermates (WT) were studied. To block the activation of NF-{kappa}B, TNF TG mice were crossed with knockout mice in which the p50 subunit of NF-{kappa}B was disrupted (p50–/– KO) [21]. Since the genetic background of TNF TG mice (FVB) was different from that of p50–/– KO mice (B6, 129), p50–/– KO mice were first backcrossed onto the FVB strain for five generations. Then, p50+/– KO males (F5) were mated with TNF TG females to yield WT or TG mice with p50+/+ or p50+/– (F6). Subsequently, TG/p50+/– females were mated with WT/p50+/– males to obtain TG or WT mice with p50+/+, p50+/–, or p50–/– (F7), which were studied in the present study. Littermates were studied in each analysis to assure the minimization of genetic background variation. All the mice studied were males unless mentioned otherwise. This study was reviewed by the Committee of the Ethics on Animal Experiment, Kyushu University Graduate School of Medical Sciences and carried out under the control of the Guideline for Animal Experiment, Kyushu University and the Law (No. 105) and Notification (No. 6) of the Government. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2. Electrophoretic mobility shift assay
Electrophoretic mobility shift assays (EMSA) were performed according to the manufacturer's instructions (Gel Shift Assay System E3300, Promega, Madison, Wisconsin, USA) as previously described [22]. Nuclear proteins were isolated using the method of Haudek [23]. Protein concentrations were measured by BCA Protein Assay Reagents (PIERCE, Rockford, Illinois, USA) using bovine serum albumin (BSA) as a standard. Protein-DNA binding was carried out in a final volume of 40 µl. To each tube, 4 µl of 10 x binding buffer (100 mmol/l Tris pH 8.0, 10 mmol/l EDTA, 40% glycerol, 1 mol/l NaCl), 100 ng of 1,4-dithiothreitol (DTT), 4 µg of BSA, 2 µg of dIdC, and 30 µg nuclear proteins were added. After the samples were incubated at room temperature for 10 min, 1 µl of 32P-labeled NF-{kappa}B probe (a double-stranded oligonucleotide corresponding to the consensus NF-{kappa}B binding site of the {kappa} light-chain enhancer: 5'-AGTTGAGGGGACTTTCCCAGGC-3', approximately 20000 cpm/ng) was added to each reaction and incubate for 20 min at room temperature. For supershift reactions, 1 µl of anti-p50 or p65 antibody (sc-114X or sc-472X: Santa Cruz, Paso Robles, California, USA) was added after 20 min binding reaction with further incubation of 30 min on ice. Samples were resolved on a 5% acrylamide gel in 0.25% TBE buffer.

2.3. Echocardiography
Echocardiographic studies were performed using an ultrasonographic system (ALOKA SSD-5500; Tokyo, Japan) as previously described [13]. Under anesthesia with 2.5% Avertin (14 µl/g body weight, IP, Aldrich Chemical), mice were placed in a supine position. A 10-MHz transducer (ALOKA) was applied to the left hemithorax. Two-dimensional targeted M-mode imaging was obtained from the short axis view at the level of the greatest LV dimension. M-mode measurements of LV end-diastolic diameter (EDD), LV end-systolic diameter (ESD), and LV anterior and posterior wall thicknesses were made using the leading edge convention of the American Society of Echocardiography. End diastole was determined at the maximal LV diastolic dimension, and end systole was taken at the peak of posterior wall motion. The percentage of LV fractional shortening (FS) was calculated as follows: FS (%)=(EDD–ESD)/EDD x 100 [13].

2.4. Pathological analysis
After measurement of body and heart weight, tissues were fixed in 10% neutral buffered formalin for hematoxylin and eosin staining or snap-frozen in liquid nitrogen for RNA and protein analysis. Cross-sectional area of cardiomyocytes in the left ventricle was evaluated as previously reported [24]. Myocardial infiltration was quantified by determination of nuclear density (nuclei/mm2) [14]. Our previous study using immunohistochemical analysis has demonstrated that most of the infiltrating cells in TNF TG mice are macrophages and CD-4-positive lymphocytes [25]. Because it is difficult to differentiate inflammatory cells from myocytes and/or fibroblasts, by simple hematoxylin and eosin staining, all nuclei were included in the present study. In each mice, five independent high-power fields were analyzed and averaged.

2.5. RNase protection assay
Total RNA was extracted from the left ventricle by an acid guanidium thiocyanate–phenolchloroform method. Multi-probe RNase protection assays (RPA) were performed according to the manufacturer's protocol (RiboQuant, PharMingen, San Diego, California, USA) using mMMP-1 (No. 551276), and a custom template set containing probes for murine RANTES, TNF-{alpha}, IL-6, IL-1β, TGF-β, monocyte chemotactic protein-1 (MCP-1), L32, and GAPDH (No. 557310) [14]. The value of each hybridized probe was normalized to that of GAPDH included in each template set as an internal control.

2.6. MMP zymography
Gelatin zymography was performed as previously described [26]. The myocardial samples were homogenized (~30-s bursts) in 1 ml of an ice-cold extraction buffer containing cacodylic acid (10 mmol/l), NaCl (0.15 mol/l), ZnCl2 (20 mmol/l), NaN2 (1.5 mmol/l), and 0.01% Triton X-100 (pH 5.0). The homogenate was then centrifuged (4 °C, 10 min, 10,000 x g), and the supernatant was decanted and saved on ice. The pH levels of the samples were adjusted to 7.5 using Tris (1 mol/l). The final protein concentration of the myocardial extract was determined using a standardized colorimetric assay. The extracted samples were then aliquoted and stored at –80 °C until the time of assay. The myocardial extracts were then directly loaded onto electrophoretic gels (SDS-PAGE) containing 1 mg/ml gelatin under nonreducing conditions. The myocardial extracts at a final protein content of 5 µg were loaded onto the gels using a 3:1 sample buffer (10% SDS, 4% sucrose, 0.25 mol/l Tris–HCl, and 0.1% bromophenol blue; pH 6.8). The gels were run at 15 mA/gel through the stacking phase (4%) and at 20 mA/gel for the separating phase (10%) while the running buffer temperature was maintained at 4 °C. After SDS-PAGE, the gels were washed twice in 2.5% Triton X-100 for 30 min each, rinsed in water, and incubated for 24 h in a substrate buffer at 37 °C (50 mmol/l Tris–HCl, 5 mmol/l CaCl2, and 0.02% NaN3; pH 7.5). After incubation, the gels were stained with Coomassie brilliant blue R-250. The zymograms were digitized, and the size-fractionated bands, which indicated the MMP proteolytic levels, were measured by the integrated optical density in a rectangular region of interest.

2.7. Statistical analysis
The results are presented as mean ± S.D. Statistical comparisons were performed using ANOVA with Student's–Newman–Keuls post-hoc test or unmatched Student's t-test where appropriate. Survival analysis was performed by the Kaplan–Meier method, and between-group difference in survival was tested by the log-rank test. Differences were considered significant at a value of P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. NF-{kappa}B KO abolishes activation of NF-{kappa}B in TNF TG myocardium
NF-{kappa}B was activated in the myocardium of TNF TG/p50+/+ mice, while it was completely abolished in TNF TG/p50–/– mice (Fig. 1a). Since most of NF-{kappa}B band was super-shifted with the anti-p50 antibody but not with the anti-p65 antibody, the majority of NF-{kappa}B was suggested to be p50–p50 homodimers in TNF TG/p50+/+ mice (Fig. 1b).


Figure 1
View larger version (42K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Electrophoretic mobility shift assay for myocardial NF-{kappa}B activation. Nuclear proteins were isolated from wild-type (WT) or TNF-{alpha} transgenic mice (TG) with or without p50 gene (a). Super-shift analysis was performed with anti-p50 or p65 antibody to investigate the composition of activated NF-{kappa}B in TG/p50+/+ mice (b).

 
3.2. NF-{kappa}B KO improves the survival of TNF TG males
Consistent with previous studies [12,13], mortality was significantly lower in female TNF TG/p50+/+ mice when compared with male TNF TG/p50+/+ mice (P<0.005, Fig. 2). Thus, NF-{kappa}B KO did not affect the survival of TNF TG females as only one of 86 TG/p50+/+, three of 137 TG/p50+/–, and four of 61 TG/p50–/– died by the end of 12 weeks (P=0.108). In contrast, NF-{kappa}B KO significantly improved the survival of TNF TG males (P<0.01) as seven of 48 TG/p50+/+ and 15 of 117 TG/p50+/–, but none of 65 TG/p50–/–, died by the end of 12 weeks. All the mice that died spontaneously exhibited exceptional dilatation of the heart and pleural effusion, suggesting that they died of congestive heart failure [11]. To elucidate the mechanisms by which NF-{kappa}B improves the survival of TNF TG mice, the following studies were focused on TNF TG males KO with p50+/+ or p50–/– at the age of 6 weeks.


Figure 2
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Kaplan–Meier survival curves of TNF-{alpha} transgenic mice: males (a) or females (b) with or without p50 gene (p50+/+, p50+/–, p50–/–). *p<0.01 vs. TG/p50+/+ mice.

 
3.3. NF-{kappa}B KO suppresses cardiac hypertrophy without ameliorating myocardial inflammation
As shown in Fig. 3a, the left ventricle of TG mice weighted significantly more than that of WT mice as previously reported [11]. NF-{kappa}B KO significantly attenuated ventricular hypertrophy in TNF TG males. After hematoxylin–eosin staining, cross-sectional area of cardiomyocytes was evaluated: WT/p50+/+ (n=3), 234 ± 24 (S.D.) µm2; WT/p50–/– (2), 245 ± 50; TNF TG/p50+/+ (5), 260 ± 70; TNF TG/p50–/– (5), 195 ± 29 (p=0.27). Despite the attenuation of ventricular hypertrophy by NF-{kappa}B KO, differences in cross-sectional were not statistically significant among the four groups. Since we have previously reported that the cardiomyocyte hypertrophy in TNF TG mice was primarily attributable to the elongation of the resting cell length without widening of the cell width [27], the attenuation of ventricular hypertrophy by NF-{kappa}B KO may be attributable to suppression of cardiomyocyte elongation in TNF TG mice. As shown in Fig. 3b, a marked infiltration of inflammatory cells was observed in the myocardium of TNF TG mice. The number of cells in the myocardium was significantly increased in TNF TG mice, although it was not affected by NF-{kappa}B KO (Fig. 3c). No significant difference was observed between TG/p50+/+ and TG/p50–/– mice.


Figure 3
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Pathological analysis of the heart: left ventricular weight (a), hematoxylin–eosin staining (b), and cell density (c). Values are mean ± S.D. WT indicates wild-type mice; TG, TNF-{alpha} transgenic mice. *p<0.05 vs. WT/p50+/+ mice, {dagger}P<0.05 vs. TG/p50+/+ mice.

 
To further assess the severity of myocardial inflammation, expression of proinflammatory cytokines was evaluated by multi-probe RPA (Fig. 4). Transcript levels of IL-1β, TGF-β, MCP-1, and RANTES as well as TNF-{alpha} were significantly increased in TNF TG myocardium. Since no significant differences were observed between TG/p50+/+ and TG/p50–/– mice, NF-{kappa}B KO did not attenuate myocardial expression of proinflammatory cytokines. These results indicate that NF-{kappa}B KO ameliorates myocardial hypertrophy without affecting myocardial inflammation in TNF TG mice.


Figure 4
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Multi-probe RNase protection assay for cytokines in myocardium: representative images (a) and summarized data (b). Values are mean ± S.D. WT indicates wild-type mice; TG, TNF-{alpha} transgenic mice. *P<0.05 vs. WT/p50+/+ mice.

 
3.4. NF-{kappa}B KO reverses cardiac dysfunction and remodeling
Echocardiography was performed to evaluate cardiac function and remodeling in these mice (Fig. 5a). TNF TG/p50+/+ mice exhibited ventricular dilatation with reduced fractional shortening. As summarized in Fig. 5b and Table 1, NF-{kappa}B KO significantly ameliorated ventricular dilatation and improved the fractional shortening in TNF TG mice. These results suggest that, despite persistent expression of proinflammatory cytokines in the myocardium, NF-{kappa}B KO improves cardiac function and restrains ventricular remodeling in TNF TG mice.


Figure 5
View larger version (42K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Echocardiographic analysis of ventricular structure and function: representative images of M-mode echocardiogram (a) and summarized data (b). Values are mean ± S.D. WT indicates wild-type mice; TG, TNF-{alpha} transgenic mice; EDD, end-diastolic diameter; ESD, end-systolic diameter; FS, fractional shortening. *p<0.05 vs. WT/p50+/+ mice, {dagger}P<0.05 vs. TG/p50+/+ mice.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Echocardiographic parameters

 
3.5. NF-{kappa}B KO blocks MMP-9 activation
Transcript levels of MMPs and TIMPs were evaluated by multi-probe RPA (Fig. 6a). MMP-2, MMP-9, and TIMP-1 were increased, while TIMP-4 was decreased, in TNF TG/p50+/+ myocardium. As summarized in Fig. 6b, NF-{kappa}B KO significantly suppressed the up-regulation of MMP-9 in TNF TG mice, although it did not affect that of MMP-2 or TIMP1, or the down-regulation of TIMP-4. To take into account of the interactions between MMPs and TIMPs in these mice, the ratios of MMP-2/TIMP-2 and MMP-9/TIMP-1 were summarized in Table 2. Although there was a trend of higher MMP-2/TIMP-2 in TNF TG mice, the up-regulation of MMP-2 was not statistically significant after normalization with TIMP-2. In contrast, although both MMP-9 and TIMP-1 were up-regulated in TNF TG mice, the ratio of MMP-9/TIMP-1 was still significantly higher in TNF TG mice. As in the case of MMP-9, NF-{kappa}B KO significantly abrogated the up-regulation of MMP-9/TIMP-1 ratio in TNF TG mice. To further confirm the specific abrogation of MMP-9 activity in TNF TG/p50–/– mice, zymography was performed as shown in Fig. 7. Although a slight but significant increase of MMP-2 activity in TNF TG mice was not affected, the marked increase of MMP-9 activity was significantly attenuated by NF-{kappa}B KO. Since we have previously reported that treatment with an MMP inhibitor significantly prolongs the survival of TNF TG mice [28], the selective inhibition of MMP-9 activation may contribute to cardioprotective effects of NF-{kappa}B KO on these mice.


Figure 6
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Multi-probe RNase protection assay for MMPs and TIMPs in myocardium: representative images (a) and summarized data (b). Values are mean ± S.D. WT indicates wild-type mice; TG, TNF-{alpha} transgenic mice. *p<0.05 vs. WT/p50+/+ mice, {dagger}P<0.05 vs. TG/p50+/+ mice.

 

View this table:
[in this window]
[in a new window]

  		Table 2 Ratios of MMPs to TIMPs

 

Figure 7
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Gelatin zymography for MMP-2 and MMP-9 in myocardium: representative images (a) and summarized data (b). Values are mean ± S.D. (n=4 each). WT indicates wild-type mice; TG, TNF-{alpha} transgenic mice. *p<0.05 vs. WT/p50+/+ mice, {dagger}P<0.05 vs. TG/p50+/+ mice.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, we tested the hypothesis that cardiotoxic effects of proinflammatory cytokines are mediated by the activation of NF-{kappa}B. Transgenic mice with cardiac-specific overexpression of TNF-{alpha} [11] were used as a model of cytokine-induced cardiomyopathy and knockout mice deficient in the p50 subunit of NF-{kappa}B [21] were used to block the activation of NF-{kappa}B. The results indicated that cardiac-specific overexpression of TNF-{alpha} provoked heart failure with NF-{kappa}B activation, myocardial inflammation, ventricular hypertrophy, cardiac dysfunction, and MMP activation. Although blockade of NF-{kappa}B did not affect myocardial inflammation, it significantly reversed ventricular hypertrophy, improved cardiac function, reduced MMP-9 activity, and improved the survival of TNF-{alpha} transgenic mice. Activation of NF-{kappa}B therefore may play an important role in the pathogenesis of myocardial dysfunction and remodeling besides promoting myocardial inflammation.

Since activation of NF-{kappa}B induces various members of proinflammatory cytokines and chemokines [17], and blockade of NF-{kappa}B activation has been shown to reduce inflammatory response and damage after cardiac ischemia and reperfusion [29], we had hypothesized that the blockade of NF-{kappa}B might attenuate myocardial inflammation observed in TNF TG mice. However, the results did not support our hypothesis: the blockade of NF-{kappa}B activation did not affect myocardial expression of proinflammatory cytokines or chemokines, or infiltration of inflammatory cells in TG myocardium. Since we have previously reported that blockade of TNF-{alpha} abrogates myocardial inflammation of TNF TG mice [14], induction of proinflammatory cytokines and chemokines and infiltration of inflammatory cells observed in TNF TG mice are provoked by TNF-{alpha}-dependent but NF-{kappa}B-independent pathways, including ceramide, ERK, phospholipase A2, and JNK [1].

The NF-{kappa}B/Rel family consists of five subunit members, including p50, p52, c-Rel, RelA (p65), and RelB [17]. In most cells, NF-{kappa}B is a heterodimer of p50 and p65 that is retained in the cytoplasm bound to the inhibitory protein I{kappa}B. Activation of NF-{kappa}B will occur when the specific I{kappa}B kinases phosphorylate the I{kappa}B. After chronic exposure to proinflammatory cytokines, including TNF-{alpha}, NF-{kappa}B has been shown to be converted from transcriptionally active p50–p65 heterodimers to transcriptionally inactive p50–p50 homodimers, which may act as a native negative feedback mechanism to prevent excessive inflammatory responses [23,30]. Since most of NF-{kappa}B band was super-shifted with the anti-p50 antibody but not with the anti-p65 antibody, the majority of NF-{kappa}B was suggested to be transcriptionally inactive p50–p50 homodimers in our TNF TG/p50+/+ mice. This might explain why the targeted disruption of the p50 subunit did not affect myocardial expression of proinflammatory cytokines in this mouse model of cytokine-induced cardiomyopathy. NF-{kappa}B-independent pathways appear to be more important in the development of myocardial inflammation with persistent overexpression of TNF-{alpha}.

Although the blockade of NF-{kappa}B did not affect myocardial inflammation, it significantly inhibited ventricular hypertrophy and dilatation, and improved the survival of TNF TG mice. Recent in vitro studies [19,20] have demonstrated that the activation of NF-{kappa}B is required for hypertrophic growth of primary rat neonatal ventricular cardiomyocytes in response to G-protein-coupled receptor agonists, including phenylephrine, endothelin-1, and angiotensin II. The results of the present study suggest that the activation of NF-{kappa}B may also play an important role in the development of hypertrophy in response to TNF-{alpha}. Although the precise mechanisms by which NF-{kappa}B mediates cardiac hypertrophy remain undetermined, it is of interest that the p50 subunit of NF-{kappa}B interacts with the transcription factor Krüppel-like factor 5 [31], an essential regulator of cardiovascular remodeling as manifested by a hypertrophic response to angiotensin II [32]. Therefore, the p50 is not only a subunit of NF-{kappa}B but may also interact with other transcription factors to exert versatile effects on cardiac hypertrophy and remodeling.

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the extracellular matrix components [33]. MMPs are increased in the failing human heart, and may play an important role in the process of cardiac remodeling [34]. We have previously reported that progressive ventricular hypertrophy and dilation in TNF TG mice are accompanied by a significant increase in MMP-2 and MMP-9 activity, an increase in collagen synthesis, deposition, and denaturation, and a decrease in undenatured collagens [15]. Furthermore, treatment with the MMP inhibitor BB-94 significantly reduces ventricular fibrosis and hypertrophy, and prolongs the survival of TNF TG mice [28]. In the present study, NF-{kappa}B KO selectively abrogated induction of MMP-9 in TNG TG myocardium. Since targeted deletion of MMP-9 has been shown to attenuate left ventricular enlargement and collagen accumulation after experimental myocardial infarction [35], the selective inhibition of MMP-9 might contribute to cardioprotective effects of NF-{kappa}B KO on TNF TG mice.

In the present study, we have demonstrated that the blockade of NF-{kappa}B activation prevents ventricular hypertrophy and remodeling in TNF-{alpha}-induced cardiomyopathy. However, the beneficial effects of NF-{kappa}B KO were limited to male TNF TG mice. The better survival function of female TNF TG mice might mask the beneficial effects of NF-{kappa}B KO. Gender difference in the survival of TNF TG mice has been repeatedly observed in our previous studies [12,13]. Echocardiographic analysis has demonstrated that impairment of baseline myocardial contractility and attenuation of β-adrenergic inotropic responsiveness are less severe in female TG mice [12]. Since the extent of myocardial expression of TNF-{alpha} has been shown to be comparable in both sexes, the gender difference is probably due to lower expression of TNF receptors in the myocardium of female TG mice [12]. Although we had hypothesized that activation of NF-{kappa}B might be less in female TG mice, our preliminary study suggests no apparent gender difference in the activation of NF-{kappa}B (data not shown). If the activation of NF-{kappa}B is similar between male and female TG mice, mechanisms by which female TG mice are protected from toxic effects of NF-{kappa}B activation remain to be determined. The role of NF-{kappa}B may be different between males and females.

Nonetheless, an inhibition of NF-{kappa}B may be a promising therapeutic strategy for cardiac remodeling and heart failure, especially when proinflammatory cytokines are activated. However, it might be undesirable to adopt systemic inhibition of NF-{kappa}B when we consider clinical application. Although NF-{kappa}B (p50) KO mice we used in the present study show no developmental abnormalities, they exhibit multifocal defects in immune responses involving B lymphocytes and nonspecific responses to infection; B cells do not proliferate in response to bacterial lipopolysaccharide and are defective in basal and specific antibody production [21]. Furthermore, inhibition of NF-{kappa}B activation in macrophages has been shown to increase atherosclerosis in LDL receptor-deficient mice, suggesting that systemic inhibition of NF-{kappa}B may promote vascular injury and atherosclerosis [36]. Although we did not detect any adverse events or premature death as long as we observed, systemic inhibition of NF-{kappa}B may be deleterious in the long run. Therefore, it might be better to develop a new technique to ensure cardiac-specific inhibition of NF-{kappa}B.

In conclusion, targeted disruption of the p50 subunit of NF-{kappa}B did not ameliorate myocardial inflammation but improved cardiac function and survival in male TNF TG mice. An inhibition of NF-{kappa}B may be a new therapeutic strategy for cardiac remodeling and heart failure, especially when proinflammatory cytokines are activated.


   Acknowledgement
 
A part of this study was conducted in Kyushu University Station for Collaborative Research. This study was supported by a grant from Kimura Memorial Heart Foundation, by the Grant for Research on Cardiovascular Disease from Japan Heart Foundation/Pfizer Pharmaceuticals Inc., and by the Grant-in-Aid for Scientific Research from the Japan Society for the promotion of Science (C15590755).


   Notes
 
Time for primary review 23 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Wang H., Czura C., Tracey K. The cytokine handbook, 4th ed. Thomson A., Lotze M., eds. (2003) San Diego: Academic Press. 837–860.
  2. Levine B., Kalman J., Mayer L., Fillit H.M., Packer M. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med (1990) 323:236–241.[Abstract]
  3. Kubota T., McNamara D.M., Wang J.J., Trost M., McTiernan C.F., Mann D.L., et al. Effects of tumor necrosis factor gene polymorphisms on patients with congestive heart failure. VEST investigators for TNF genotype analysis. Vesnarinone survival trial. Circulation (1998) 97:2499–2501.[Abstract/Free Full Text]
  4. Torre-Amione G., Kapadia S., Lee J., Durand J.B., Bies R.D., Young J.B., et al. Tumor necrosis factor-alpha and tumor necrosis factor receptors in the failing human heart. Circulation (1996) 93:704–711.[Abstract/Free Full Text]
  5. Kubota T., Miyagishima M., Alvarez R.J., Kormos R., Rosenblum W.D., Demetris A.J., et al. Expression of proinflammatory cytokines in the failing human heart: comparison of recent-onset and end-stage congestive heart failure. J Heart Lung Transplant (2000) 19:819–824.[CrossRef][Web of Science][Medline]
  6. Finkel M.S., Oddis C.V., Jacob T.D., Watkins S.C., Hattler B.G., Simmons R.L. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science (1992) 257:387–389.[Abstract/Free Full Text]
  7. Yokoyama T., Vaca L., Rossen R.D., Durante W., Hazarika P., Mann D.L. Cellular basis for the negative inotropic effects of tumor necrosis factor-a in the adult mammalian heart. J Clin Invest (1993) 92:2012–2303.
  8. Yokoyama T., Nakano M., Bendnarczyk J.L., McIntyre B.W., Entman M., Mann D.L. Tumor necrosis factor-a provokes a hypertrophic growth response in adult cardiac myocytes. Circulation (1997) 95:1247–1252.[Abstract/Free Full Text]
  9. Nakamura K., Fushimi K., Kouchi H., Mihara K., Miyazaki M., Ohe T., et al. Inhibitory effects of antioxidants on neonatal rat cardiac myocyte hypertrophy induced by tumor necrosis factor-alpha and angiotensin II. Circulation (1998) 98:794–799.[Abstract/Free Full Text]
  10. Krown K.A., Page M.T., Nguyen C., Zechner D., Gutierrez V., Comstock K.L., et al. Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes. Involvement of the sphingolipid signaling cascade in cardiac cell death. J Clin Invest (1996) 98:2854–2865.[Web of Science][Medline]
  11. Kubota T., McTiernan C.F., Frye C.S., Slawson S.E., Lemster B.H., Koretsky A.P., et al. Dilated cardiomyopathy in transgenic mice with cardiac-specific overexpression of tumor necrosis factor-alpha. Circ Res (1997) 81:627–635.[Abstract/Free Full Text]
  12. Kadokami T., McTiernan C.F., Kubota T., Frye C.S., Feldman A.M. Sex-related survival differences in murine cardiomyopathy are associated with differences in TNF-receptor expression. J Clin Invest (2000) 106:589–597.[Web of Science][Medline]
  13. Funakoshi H., Kubota T., Kawamura N., Machida Y., Feldman A.M., Tsutsui H., et al. Disruption of inducible nitric oxide synthase improves beta-adrenergic inotropic responsiveness but not the survival of mice with cytokine-induced cardiomyopathy. Circ Res (2002) 90:959–965.[Abstract/Free Full Text]
  14. Kubota T., Bounoutas G.S., Miyagishima M., Kadokami T., Sanders V.J., Bruton C., et al. Soluble tumor necrosis factor receptor abrogates myocardial inflammation but not hypertrophy in cytokine-induced cardiomyopathy. Circulation (2000) 101:2518–2525.[Abstract/Free Full Text]
  15. Li Y.Y., Feng Y.Q., Kadokami T., McTiernan C.F., Draviam R., Watkins S.C., et al. Myocardial extracellular matrix remodeling in transgenic mice overexpressing tumor necrosis factor alpha can be modulated by anti-tumor necrosis factor alpha therapy. Proc Natl Acad Sci U S A (2000) 97:12746–12751.[Abstract/Free Full Text]
  16. Bryant D., Becker L., Richardson J., Shelton J., Franco F., Peshock R., et al. Cardiac failure in transgenic mice with myocardial expression of tumor necrosis factor-alpha. Circulation (1998) 97:1375–1381.[Abstract/Free Full Text]
  17. Barnes P.J., Karin M. Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med (1997) 336:1066–1071.[Free Full Text]
  18. Purcell N.H., Molkentin J.D. Is nuclear factor kappaB an attractive therapeutic target for treating cardiac hypertrophy? Circulation (2003) 108:638–640.[Free Full Text]
  19. Purcell N.H., Tang G., Yu C., Mercurio F., DiDonato J.A., Lin A. Activation of NF-kappa B is required for hypertrophic growth of primary rat neonatal ventricular cardiomyocytes. Proc Natl Acad Sci U S A (2001) 98:6668–6673.[Abstract/Free Full Text]
  20. Hirotani S., Otsu K., Nishida K., Higuchi Y., Morita T., Nakayama H., et al. Involvement of nuclear factor-kappaB and apoptosis signal-regulating kinase 1 in G-protein-coupled receptor agonist-induced cardiomyocyte hypertrophy. Circulation (2002) 105:509–515.[Abstract/Free Full Text]
  21. Sha W.C., Liou H.C., Tuomanen E.I., Baltimore D. Targeted disruption of the p50 subunit of NF-kappa B leads to multifocal defects in immune responses. Cell (1995) 80:321–330.[CrossRef][Web of Science][Medline]
  22. Kubota T., Miyagishima M., Frye C.S., Alber S.M., Bounoutas G.S., Kadokami T., et al. Overexpression of tumor necrosis factor-alpha activates both anti- and pro-apoptotic pathways in the myocardium. J Mol Cell Cardiol (2001) 33:1331–1344.[CrossRef][Web of Science][Medline]
  23. Haudek S.B., Bryant D.D., Giroir B.P. Differential regulation of myocardial NF kappa B following acute or chronic TNF-alpha exposure. J Mol Cell Cardiol (2001) 33:1263–1271.[CrossRef][Web of Science][Medline]
  24. Shiomi T., Tsutsui H., Ikeuchi M., Matsusaka H., Hayashidani S., Suematsu N., et al. Streptozotocin-induced hyperglycemia exacerbates left ventricular remodeling and failure after experimental myocardial infarction. J Am Coll Cardiol (2003) 42:165–172.[Abstract/Free Full Text]
  25. Machida Y., Kubota T., Kawamura N., Funakoshi H., Ide T., Utsumi H., et al. Overexpression of tumor necrosis factor-alpha increases production of hydroxyl radical in murine myocardium. Am J Physiol Heart Circ Physiol (2003) 284:H449–H455.[Abstract/Free Full Text]
  26. Hayashidani S., Tsutsui H., Ikeuchi M., Shiomi T., Matsusaka H., Kubota T., et al. Targeted deletion of MMP-2 attenuates early LV rupture and late remodeling after experimental myocardial infarction. Am J Physiol Heart Circ Physiol (2003) 285:H1229–H1235.[Abstract/Free Full Text]
  27. Janczewski A.M., Kadokami T., Lemster B., Frye C.S., McTiernan C.F., Feldman A.M. Morphological and functional changes in cardiac myocytes isolated from mice overexpressing TNF-alpha. Am J Physiol Heart Circ Physiol (2003) 284:H960–H969.[Abstract/Free Full Text]
  28. Li Y.Y., Kadokami T., Wang P., McTiernan C.F., Feldman A.M. MMP inhibition modulates TNF-alpha transgenic mouse phenotype early in the development of heart failure. Am J Physiol Heart Circ Physiol (2002) 282:H983–H989.[Abstract/Free Full Text]
  29. Morishita R., Sugimoto T., Aoki M., Kida I., Tomita N., Moriguchi A., et al. In vivo transfection of cis element "decoy" against nuclear factor-kappaB binding site prevents myocardial infarction. Nat Med (1997) 3:894–899.[CrossRef][Web of Science][Medline]
  30. Lawrence T., Gilroy D.W., Colville-Nash P.R., Willoughby D.A. Possible new role for NF-kappaB in the resolution of inflammation. Nat Med (2001) 7:1291–1297.[CrossRef][Web of Science][Medline]
  31. Aizawa K., Suzuki T., Kada N., Ishihara A., Kawai-Kowase K., Matsumura T., et al. Regulation of platelet-derived growth factor-A chain by Krüppel-like factor 5: new pathway of cooperative activation with nuclear factor-kappaB. J Biol Chem (2004) 279:70–76.[Abstract/Free Full Text]
  32. Shindo T., Manabe I., Fukushima Y., Tobe K., Aizawa K., Miyamoto S., et al. Kruppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin II signaling and an essential regulator of cardiovascular remodeling. Nat Med (2002) 8:856–863.[Web of Science][Medline]
  33. Spinale F.G. Matrix metalloproteinases: regulation and dysregulation in the failing heart. Circ Res (2002) 90:520–530.[Abstract/Free Full Text]
  34. Li Y.Y., Feldman A.M., Sun Y., McTiernan C.F. Differential expression of tissue inhibitors of metalloproteinases in the failing human heart. Circulation (1998) 98:1728–1734.[Abstract/Free Full Text]
  35. Ducharme A., Frantz S., Aikawa M., Rabkin E., Lindsey M., Rohde L.E., et al. Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest (2000) 106:55–62.[Web of Science][Medline]
  36. Kanters E., Pasparakis M., Gijbels M.J., Vergouwe M.N., Partouns-Hendriks I., Fijneman R.J., et al. Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor-deficient mice. J Clin Invest (2003) 112:1176–1185.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Circ. Res.Home page
S. Hikoso, O. Yamaguchi, Y. Nakano, T. Takeda, S. Omiya, I. Mizote, M. Taneike, T. Oka, T. Tamai, J. Oyabu, et al.
The I{kappa}B Kinase {beta}/Nuclear Factor {kappa}B Signaling Pathway Protects the Heart From Hemodynamic Stress Mediated by the Regulation of Manganese Superoxide Dismutase Expression
Circ. Res., July 2, 2009; 105(1): 70 - 79.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. X. L. Liu, R. Tian, H. Baskind, W. Hsueh, and I. G. De Plaen
Platelet-activating factor induces the processing of nuclear factor-{kappa}B p105 into p50, which mediates acute bowel injury in mice
Am J Physiol Gastrointest Liver Physiol, July 1, 2009; 297(1): G76 - G81.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
S. Frantz and J. Bauersachs
Regarding the Article by Timmers et al
Circ. Res., June 5, 2009; 104(11): e60 - e60.
[Full Text] [PDF]

Home page
 Circ. Res.Home page
L. Timmers and D. P.V. de Kleijn
Response to the Letter by Frantz and Bauersachs
Circ. Res., June 5, 2009; 104(11): e61 - e61.
[Full Text] [PDF]

Home page
 Circ. Res.Home page
L. Timmers, J. K. van Keulen, I. E. Hoefer, M. F.L. Meijs, B. van Middelaar, K. den Ouden, C. J.A. van Echteld, G. Pasterkamp, and D. P.V. de Kleijn
Targeted Deletion of Nuclear Factor {kappa}B p50 Enhances Cardiac Remodeling and Dysfunction Following Myocardial Infarction
Circ. Res., March 13, 2009; 104(5): 699 - 706.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
F. G. Spinale
Myocardial Matrix Remodeling and the Matrix Metalloproteinases: Influence on Cardiac Form and Function
Physiol Rev, October 1, 2007; 87(4): 1285 - 1342.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
S. Frantz, J. Tillmanns, P. J. Kuhlencordt, I. Schmidt, A. Adamek, C. Dienesch, T. Thum, S. Gerondakis, G. Ertl, and J. Bauersachs
Tissue-Specific Effects of the Nuclear Factor {kappa}B Subunit p50 on Myocardial Ischemia-Reperfusion Injury
Am. J. Pathol., August 1, 2007; 171(2): 507 - 512.
[Abstract] [Full Text] [PDF]

Home page
 J Am Coll CardiolHome page
D. Hilfiker-Kleiner, U. Landmesser, and H. Drexler
Molecular Mechanisms in Heart Failure: Focus on Cardiac Hypertrophy, Inflammation, Angiogenesis, and Apoptosis
J. Am. Coll. Cardiol., October 27, 2006; 48(9_Suppl_A): A56 - A66.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
S. Frantz, K. Hu, B. Bayer, S. Gerondakis, J. Strotmann, A. Adamek, G. Ertl, and J. Bauersachs
Absence of NF-{kappa}B subunit p50 improves heart failure after myocardial infarction
FASEB J, September 1, 2006; 20(11): 1918 - 1920.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. Kawano, T. Kubota, Y. Monden, T. Tsutsumi, T. Inoue, N. Kawamura, H. Tsutsui, and K. Sunagawa
Blockade of NF-{kappa}B improves cardiac function and survival after myocardial infarction
Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1337 - H1344.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
A. M. Deschamps and F. G. Spinale
Pathways of matrix metalloproteinase induction in heart failure: Bioactive molecules and transcriptional regulation
Cardiovasc Res, February 15, 2006; 69(3): 666 - 676.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Y. Higuchi, T. O. Chan, M. A. Brown, J. Zhang, B. R. DeGeorge Jr., H. Funakoshi, G. Gibson, C. F. McTiernan, T. Kubota, W. K. Jones, et al.
Cardioprotection afforded by NF-{kappa}B ablation is associated with activation of Akt in mice overexpressing TNF-{alpha}
Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H590 - H598.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Kawamura, N.
Right arrow Articles by Sunagawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kawamura, N.
Right arrow Articles by Sunagawa, K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?