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B subunit p50 improves heart failure after myocardial infarction
Medizinische Klinik und Poliklinik I, Herzkreislauf-Zentrum, Universität Würzburg, Germany; and
* The Walter and Eliza Hall Institute of Medical Research, Parcville, Victoria, Australia
1Correspondence: Medizinische Klinik und Poliklinik I, Universitat Würzburg, Josef-Schneider-Str. 2, Würzburg 97080, Germany. E-mail: frantz_s{at}medizin.uni-wuerzburg.de
ABSTRACT
Background: NF kappa B (NF-
B) is a ubiquitous transcription factor activated by various stimuli implicated in heart failure progression including reactive oxygen species (ROS), hypoxia, and inflammatory cytokines. Although NF-B is involved in ischemic preconditioning, unstable angina pectoris, and atherogenesis, its role in heart failure has not been determined. Therefore, we investigated left ventricular remodeling in mice with a targeted deletion of the NF-B subunit p50/NF-B1 after myocardial infarction. Methods and results: p50 knockout (KO) and wild-type (WT) animals underwent coronary artery ligation. Transthoracic echocardiography was performed at days 0, 21, and 56 at midpapillary levels. Early mortality was significantly lower in KO than in WT animals. Moreover, p50 KOs exhibited significantly reduced ventricular dilatation over 8 wk compared to WT controls (end-systolic diameters by transthoracic echocardiography, WT vs. KO, 0.55±0.04 vs. 0.34±0.03 cm) and preserved left ventricular contractility. Collagen content and matrixmetalloproteinase (MMP) -9 expression were significantly lower in KO mice after myocardial infarction and may account for improved left ventricular remodeling. Conclusions: Absence of the NF-B subunit p50 improves early survival and reduces left ventricular dilatation after myocardial infarction. NF-B might therefore be an attractive target to treat heart failure.—Frantz, S., Hu, K., Bayer, B., Gerondakis, S., Strotmann, J., Adamek, A., Ertl, G., Bauersachs, J. Absence of NF-B subunit p50 improves heart failure after myocardial infarction.
Key Words: inflammation • NF kappa B • interleukins • collagen
THE ACTIVATION OF various cytokines and Toll-like receptors has been described as part of the innate immune response in experimental and human chronic heart failure (1)
. These innate immune proteins share the downstream activation of NF kappa B (NF-B). Attenuation of NF-B signaling might therefore be an interesting new therapeutic strategy to reduce the activation of the innate immune response in congestive heart failure (CHF) with potential protective effects.
Different drugs, such as PDTC (pyrolidine dithiocarbamate), have been used to inhibit NF-B activation in experimental settings. However, none of these compounds has sufficient specificity to conclusively clarify the role of NF-B, e.g., PDTC also acts as a reactive oxygen scavenger and iron chelator. Therefore, the best possibility to specifically investigate NF-B function in experimental settings is to use mice that lack NF-B function. In the rodent heart, functional NF-B requires formation of heterodimers of the p50 and p65 subunit (2)
. The p65 knockout (KO) is embryonically lethal due to liver apoptosis. By contrast, the p50 KO mouse displays a rather mild phenotype; these mice undergo normal development but show an altered immune response that includes a defective B cell proliferation in response to lipopolysaccharides (3)
. p50 might thus be an ideal target to suppress innate immune activation and to test the hypothesis of a detrimental role for NF-B activation in CHF.
Thus, we tested the functional role of NF-B for left ventricular (LV) remodeling after myocardial infarction (MI) in mice with targeted deletion of the NF-B subunit p50.
MATERIALS AND METHODS
Animals and surgery
p50 KO mice were created and characterized by Sha et al. in 1995 (3)
. p50 mice have been backcrossed in the C57Bl/6 background for 10 generations by SG. Mice 8- to 12-wk-old with a body wt of 20–28 g underwent left coronary artery ligation to induce MI, as described (4)
. The governmental Standing Committee on Animal Research has approved the animal study protocol.
Echocardiographic analysis
Ultrasound analyses were performed by a single researcher experienced in rodent echocardiography blinded to mouse genotype at days 0, 21, and 56 immediately before sacrificing the animals, as recently described (4
, 5)
. Our intraobserver variability was 4.2% as measured over four independent variables at three different time points. All animals after MI were serially imaged. From 2-dimensional short axis imaging, endocardial borders were traced at end-systole and end-diastole utilizing a prototype off-line analysis system (NICE, Toshiba Medical Systems, The Netherlands). Measurements were performed at the midpapillary muscle level. The end-systolic (smallest) and end-diastolic (largest) cavity areas were determined. Using the end-systolic and -diastolic areas, fractional area changes were calculated [(end-diastolic area – end-systolic area)/end – diastolic area]. From 2-dimensionally targeted M-mode tracings, end-diastolic diameter and end-systolic diameter were measured. Fractional shortening was calculated. Only animals with an infarct size of >30% and a heart rate greater than 450/min were included.
Sample collection, determination of infarct size, and ventricular remodeling
The left ventricle was cut into three transverse sections: apex, middle ring, and base as previously reported (4)
. From the middle ring, 5 µm sections were stained with picrosirius red. Infarct size (fraction of the infarcted left ventricle) was calculated as the percentage of length of circumference. Animals were allocated to the sham group when there was no histological sign of myocardial infarction.
Biochemical and molecular measurements
Myocardial RNA isolation and real-time polymerase chain reaction (PCR) measurements were performed as described previously (4)
with commercially available TaqMan probes for 18S, murine TGF (transforming growth factor), IL-1ß (interleukin-1ß), TNF (tumor necrosis factor), and collagen 1 (Applied Biosystems, Foster City, CA, USA). RNA samples were normalized to 18S rRNA.
Apoptosis measurement
Caspase3/7 activity (Promega, Mannheim, Germany) and cytoplasmatic histone-associated DNA fragments (ELISA, Roche, Penzberg, Germany) were measured as recommended by the manufacturer’s protocol. Moreover, apoptosis was measured by a TUNEL assay (Roche, Penzberg, Germany) as described previously (6)
.
Collagen content
Picrosirius red polarization microscopy was performed to detect interstitial collagen according to a modified method of Junqueira. Type I and III collagen in the septum was identified under illumination with polarized light. Sections from a subset of 8 WT and 7 KO mice that survived the 56 day protocol were chosen with similar myocardial infarction sizes. Sections were visualized under polarized light, photographed with the same exposure time for each section, and collagen content was measured as described previously (4)
. MMP-9 (matrixmetalloproteinase 9) was measured in duplicate by a commercial ELISA (R&D Systems, Abingdon, UK) according to the manufacture’s protocol as described with tissue from the remote region after myocardial infarction (7)
.
Preparation of nuclear and cytosolic extract
Nuclear and cytosolic proteins were extracted from myocardium harvested 3 days after myocardial infarction (8)
. After Dounce homogenization cells were lysed for 10 min on ice in a solution containing 10 mM HEPES (pH 7.6), 10 mM KCl, 1.5 mM MgCl2, 0.5% Nonidet-40, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). Nuclei were precipitated by centrifugation at 800 g for 30 s, supernatants saved as cytosolic extracts, and the nuclei resuspended in a solution of 20 mM HEPES, 1.5 mM MgCl2, 420 mM KCl, 0.2 mM EDTA, 1 mM DTT, and 0.5 mM PMSF. The mixture was incubated on ice for 30 min, the supernatant collected after centrifugation for 15 min at 13,000 g, and an equal amount of glycerol buffer was added (20 mM HEPES, 100 mM KCl, 0.2 mM EDTA, 20% glycerol).
Electromobility shift assay (EMSA)
Electromobility shift assays were performed as described (9)
. Binding reactions were performed with 10 µg of nuclear protein. Control reaction mixtures contained a 100-fold excess of unlabeled oligonucleotide and were incubated with nuclear extracts as indicated. DNA complexes were separated on a 5% nondenaturing polyacrylamide gel in Tris-borate EDTA buffer. NF-B oligonucleotides were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA).
Immunohistochemistry
Formalin fixed sections of mouse myocardium were prepared in the standard manner. Cells were labeled by the sequential application of the primary rat anti-mouse macrophage antibody (Ab) F4/80 (Research Diagnostics Inc., Flanders, NJ, USA), a primary rat anti-mouse neutrophil Ab (Clone 7/4; Linaris, Wertheim, Germany), or a nonimmune immunoglobulin (Ig) and a peroxidase antiperoxidase complex, followed by labeling (Vectastain avidin-biotin complex (ABC) Kit, Vector Laboratories, Burlingame, CA, USA). The slides were washed, dehydrated, and mounted for light microscopy.
Statistical analysis
All replicate data are expressed as mean and SE. Mortality rates were compared using a log rank test. Absolute differences among groups were compared using a 2-way ANOVA adjusted by the Fisher rule. Statistical significance was achieved when 2-tailed P < 0.05. Statistical analyses were carried out using StatView statistics program (Abacus Concepts, Inc., Berkley, CA, USA).
RESULTS
Mortality and organ weights in mice with targeted deletion of the NF-B subunit p50 after myocardial infarction
Sixty-three animals survived coronary artery ligation and had echocardiographic and histological signs of MI (32 WT, 31 KO). Mortality was significantly higher in the WT group between day 4 and day 9 (Fig. 1
). However, this enhanced survival among the KO animals was not sustained over the time course of 8 wk. Death was suspected to be attributable to rupture (2 KO, 2WT), heart failure, and arrhythmias.
|
LV weights and markers of cardiac hypertrophy (ßMHC/
MHC) were not affected by the genotype after MI (Table 1
). Infarct size determined 8 wk after MI was comparable in both groups (wild-type vs. KO, 55.8±4.4 vs. 52.4±4.1%, P=n.s.).
|
Echocardiographic measurements in mice with targeted deletion of the NF-B subunit p50 after myocardial infarction
In an initial echocardiography before coronary artery ligation, WT and KO animals did not display significant differences (Table 2
). Animals underwent follow-up echocardiography at wk 3 and 8 after MI. All measurements were recorded at the midpapillary level, which shows changes in the dimensions of the surviving noninfarcted myocardium. Animals without echocardiographic and histological signs of MI were classified as shams. In WT animals ventricles following MI tended to progressively dilate over time, with significantly larger end-diastolic and end-systolic areas as well as diameters 3 and 8 wk after MI. However, KO mice exhibited significantly less left ventricular dilatation than WT animals (see Fig. 2
and Table 2
); in fact, papillary dimensions of infarcted KO animals were not significantly different from sham animals 8 wk after MI. In addition, fractional shortening was significantly lower in the WT group (see Fig. 2
).
|
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Activation of inflammation after myocardial infarction
Nuclear translocation of NF-B was studied by EMSA after MI. There was no significant nuclear translocation of NF-B in animals with targeted deletion of the NF-B subunit p50 in the heart (see Fig. 3
).
|
Under basal conditions, WT and KO animals had similar expression levels of myocardial TNF as measured by real-time PCR (see Table 1
). TNF expression was higher in p50 KO mice starting 1 day after myocardial infarction (see Table 1
and Fig. 4
).
|
The genotype did not influence infiltration of inflammatory cells 1 day after myocardial infarction (neutrophils, WT vs. KO, 364±44 vs. 437±114/section, P=n.s.; macrophages, WT vs. KO, 4±2 vs. 5±2/section, P=n.s.).
Apoptosis after myocardial infarction
Apoptosis was not different between the groups as measured by a caspase 3/7 activity assay (wild-type vs. KO, 2377±271 vs. 2612±435, arbitrary units, P=ns) and by TUNEL assay (wild-type vs. KO, 45±2 vs. 48±3% TUNEL-positive nuclei/nuclei, P=ns) 1 day after MI, by cytoplasmic histone-associated DNA fragments 3 days after myocardial infarction (wild-type vs. KO, 1171±351 vs. 829±238 mU/mg heart, P=ns), and by TUNEL assay 8 wk after myocardial infarction in the remote area (wild-type vs. KO, 17±2 vs. 15±1% TUNEL-positive nuclei/nuclei, P=ns).
Collagen metabolism after myocardial infarction
After MI there was a profound change in collagen deposition: mRNA expression levels of collagen 1 (infarct region, WT vs. KO, 2.1±0.3 vs. 0.9±0.4, arbitrary units, P=0.04) and total collagen content were markedly decreased in KO animals (collagen volume fraction in the septum, WT vs. KO, 1.48±0.16 vs. 0.41±0.07%, P<0.001, see Fig. 5
). MMP-9 tissue levels were significantly lower in p50 KO animals 3 days (MMP-9, WT vs. KO, 2.95±0.56 vs. 0.71±0.16 ng/10 µg protein, P<0.001, see Fig. 5
), but not 8 wk after myocardial infarction (MMP-9, WT vs. KO, 0.0±0.0 vs. 0.01±0.01 ng/10 µg protein, P=n.s.). TGF expression 3 days after myocardial infarction was not influenced by the genotype (wild-type vs. KO, 0.6±0.1 vs. 0.7±0.1 arbitrary units, P=n.s.).
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DISCUSSION
The major findings of this study are reduced early mortality and ventricular dilatation as well as preserved LV systolic function in p50-deficient animals after MI. Several mechanisms may explain the protection conferred by p50/NF-B1 deficiency. The most obvious reason would have been a reduction in the immediate immune response cytokines that are regulated by NF-B, such as TNF and interleukin (IL)-1ß, since increased production of both cytokines has been implicated in the pathophysiology of heart failure (10)
. However, we did not detect the expected reduction of myocardial expression of TNF and IL-1ß. In contrast, LV expression of both cytokines was significantly higher in the p50 KO after MI despite a profound reduction of NF-B activation in KO hearts. This is in accordance with two recent studies (11
, 12)
. Kawano et al. (11)
demonstrated reduced myocardial hypertrophy in p50 KO mice in response to angiotensin (ANG) II infusion. In parallel to our study, TNF was significantly more induced in the p50 KO mice by ANG II than in matching WT mice. Moreover, in p50 KO mice TNF does not induce NF-B activation resulting in resistance to TNF-induced cardiomyopathy (12)
. However, expression of proinflammatory proteins was not affected in double-transgenic mice with p50 KO and cardiomyocyte-specific overexpression of TNF. Thus, induction of proinflammatory proteins in response to hypertrophy, proinflammatory cytokines, or ischemia may not be mediated by NF-B (13)
. Increased cytokine levels in p50 KO mice may be related to the different transcriptional activity of p50-p65 heterodimers and p50-p50 homodimers (14)
. Chronic exposure of proinflammatory cytokines, including TNF, can shift the ratio of transcriptionally active heterodimers to the transcriptionally inactive p50 homodimers, which can act as negative feedback mechanism to prevent excessive production of proinflammatory proteins and thereby may account for the increased TNF expression in the p50 KO. Nonetheless, TNF and p50 double-transgenic mice were protected from TNF-induced cardiomyopathy, not the production of proinflammatory proteins, but their impaired signaling seems to be important for the protective effects observed.
Recent studies by several investigators have implicated NF-B activation as an important event in cardiac hypertrophy (15)
. Yet heart weights and molecular hypertrophy markers were not different between the WT and KO group in our study, suggesting that LV hypertrophy was not affected by loss of p50. Moreover, several ischemia/reperfusion studies found a reduction in infarct size when NF-B was inhibited (16)
. Although at the end of our study there was no difference in infarct sizes, our study results are limited by the fact that infarct sizes soon after myocardial infarction were not measured and that an early reduction of infarct size in the KO animals could potentially explain our results.
Low levels of myocyte apoptosis have been identified in CHF and could recently be functionally linked to the progression of heart failure (17)
. NF-B is critically involved in pro- and antiapoptotic pathways in the heart. However, in our study the rate of apoptosis as measured by three different methods was not different between WT and KO animals.
Changes in extracellular matrix (ECM) composition contribute to progressive left ventricular remodeling (18)
. Administration of a prolyl 4-hydroxylase inhibitor postmyocardial infarction prevented interstitial fibrosis and thereby reduced LV enlargement (19)
. Moreover, targeted deletion of the MMP-9 gene attenuated LV dilation after experimental MI in mice and decreased collagen accumulation (20)
. Both collagen and MMP-9 transcription are regulated by NF-B (21
, 22)
. Consequently, in our model collagen and MMP-9 expression were significantly reduced in the p50 KO mice after MI. Thus, changes in ECM composition may account for improved LV remodeling in the p50 KO.
Our results are in contrast to findings by Misra et al., who recently published an increase in infarct size and rate of apoptosis 24 h after MI in mice with cardiac specific overexpression of an inhibitor of NF-B (23)
. Reasons for this discrepancy may be that different strategies of NF-B inhibition were used. Whereas Misra et al. used a dominant negative construct, in our mouse the specific subunit p50 was deleted. Second, while Misra et al. used a cardiomyoctye-specific transgenic animal, we report here results from a global KO. Therefore, attenuation of NF-B activation in other cell types involved in myocardial remodeling, such as fibroblast or inflammatory cells, may contribute to the results reported by us.
In summary, in mice with targeted disruption of the NF-B subunit p50, early mortality after MI is reduced and ventricular dilatation is prevented, which is linked to decreased collagen production and deposition. p50 might therefore be an interesting novel target in post-MI remodeling and heart failure.
ACKNOWLEDGMENTS
This work was supported in part by the Deutsche Forschungsgemeinschaft (Fr. 1377/4-3; SFB 688 TPA10). We thank D. Baltimore for the gift of the p50 KO mice and H. Wagner and D. Fraccarollo for technical support.
Received for publication September 29, 2005. Accepted for publication April 10, 2006.
REFERENCES
This article has been cited by other articles:
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  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]
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 M. W. Bergmann and L. J. De Windt Linking Cardiac Mechanosensing at the Sarcomere M-Band, Nuclear Factor {kappa}B Signaling, and Cardiac Remodeling Hypertension, June 1, 2007; 49(6): 1225 - 1227. [Full Text] [PDF]
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0.05, WT vs. KO).
P 
