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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
SPECIFIC AIMS
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 (MI).
PRINCIPAL FINDINGS
1. Decreased early mortality after myocardial infarction in mice with targeted deletion of the NF-B subunit p50
Sixty-three animals survived coronary artery ligation and had echocardiographic and histological signs of MI (32 wild-type (WT), 31 KO). Mortality was significantly higher in the WT group between day 4 and day 9. 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 (ßbeta;MHC/
MHC) were not affected by the genotype after MI. 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.).
2. Improved left ventricular remodeling in p50 KO mice
In an initial echocardiography before coronary artery ligation, WT and KO animals did not display significant differences. 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. In WT animals ventricles after 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 when compared to WT animals (see Fig. 1
); in fact, papillary dimensions of infarcted KO animals were not significantly different from sham animals 8 wk after MI.
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3. Collagen analysis, apoptosis, and proinflammatory cytokines in p50 KO mice
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. Under basal conditions, WT and KO animals had similar expression levels of myocardial TNF as measured by real-time polymerase chain reaction (PCR). However, TNF expression was higher in p50 KO mice starting 1 day after myocardial infarction (day 56, WT vs. KO, 8.3±0.2 vs. 42.6±1.8, arbitrary units, P<0.05).
Apoptosis was not different between the groups as measured by a caspase 3/7 activity assay and by TUNEL assay 1 day after MI, by cytoplasmic histone-associated DNA fragments 3 days after myocardial infarction, and by TUNEL assay 8 wk after myocardial infarction.
However, after MI there was a profound change in collagen deposition: mRNA expression levels of collagen 1 and total collagen content were markedly decreased in KO animals (see Fig. 2
). 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. 2
), but not 8 wk after myocardial infarction.
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CONCLUSIONS AND SIGNIFICANCE
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)-1ßbeta;, since increased production of both cytokines has been implicated in the pathophysiology of heart failure. However, we did not detect the expected reduction of myocardial expression of TNF and IL-1ßbeta;. 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: Kawano et al. 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. 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. Increased cytokine levels in p50 KO mice may be related to the different transcriptional activity of p50-p65 heterodimers and p50-p50 homodimers. 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, since 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 noted.
Recent studies by several investigators have implicated NF-B activation as an important event in cardiac hypertrophy. 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. Although at the end of our study there was no difference of 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. NF-B is critically involved in pro- and antiapoptotic pathways in the heart. However, in our study the rate of apoptosis 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. Administration of a prolyl 4-hydroxylase inhibitor postmyocardial infarction prevented interstitial fibrosis and thereby reduced LV enlargement. Moreover, targeted deletion of the MMP-9 gene attenuated LV dilation following experimental MI in mice and decreased collagen accumulation. Both collagen and MMP-9 transcription are regulated by NF-B. 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.
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 and novel target in post-MI remodeling and heart failure.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5133fje
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