The IκB Kinase β/Nuclear Factor κB Signaling Pathway Protects the Heart From Hemodynamic Stress Mediated by the Regulation of Manganese Superoxide Dismutase Expression
Cardiomyocyte death plays an important role in the pathogenesis of heart failure. The nuclear factor (NF)-κB signaling pathway regulates cell death, however, the effect of NF-κB pathway on cell death can vary in different cells or stimuli. The purpose of the present study was to clarify the in vivo role of the NF-κB pathway in response to pressure overload. First, we subjected C57Bl6/J mice to pressure overload by means of transverse aortic constriction (TAC) and examined the activity of the NF-κB pathway in response to pressure overload. IκB kinase (IKK) and NF-κB were activated after TAC. Then, we investigated the role of the activation using cardiac-specific IKKβ-deficient mice (CKO). CKO displayed normal global cardiac structure and function compared with control littermates. We subjected CKO and control mice to pressure overload. One week after TAC, CKO showed cardiac dilation, dysfunction, and lung congestion, which are characteristics of heart failure. The number of apoptotic cells in the hearts of CKO mice increased significantly after TAC. The levels of manganese superoxide dismutase mRNA and protein expression in CKO after TAC were significantly attenuated compared with control mice. The levels of oxidative stress and c-Jun N-terminal kinase (JNK) activation in CKO after TAC were significantly greater than those in control mice. Isoproterenol-induced cell death of isolated adult CKO cardiomyocytes was inhibited by treatment with either a manganese superoxide dismutase mimetic or a JNK inhibitor. Thus, the IKKβ/NF-κB signaling pathway plays a protective role in cardiomyocytes because of the attenuation of oxidative stress and JNK activation in a setting of acute pressure overload.
Cardiac remodeling is generally accepted as a determinant of the clinical course of heart failure. Cardiomyocyte apoptotic death plays an important role in the progression of cardiac remodeling.1–4 The loss of cardiomyocytes caused by apoptosis is predicted to reduce contractility and promote slippage of muscle bundles, wall thinning, and dilatation, which are commonly observed during heart failure. Neurohumoral factors and cytokines that are induced by mechanical stress on cardiomyocytes activate various intracellular signaling pathways, which regulate apoptotic cell death.
The nuclear factor (NF)-κB transcription factors (p50, p52, RelA, c-Rel, and RelB) play important roles in many physiological and pathological conditions. These transcriptional factors are kept inactive in the cytoplasm by binding of inhibitory proteins, the IκB (inhibitor of NF-κB) family. On stimulation, IκBs are phosphorylated at serine residues, leading to their ubiquitination and degradation by the 26S proteasome. The freed NF-κB components dimerize and translocate to nucleus, where they bind to specific sequences in either the promoter or enhancer regions of target genes.5 Activation process is dependent on phosphorylation of IκB proteins, which is mediated by the IKK complex. The IKK complex is composed of 2 catalytic subunits (IKKα and IKKβ) and a regulatory subunit (IKKγ or NEMO [NF-κB essential modulator]). In the canonical NF-κB signaling pathway, IKKβ is both necessary and sufficient for phosphorylation of IκBα and IκBβ.5
We previously reported that NF-κB is involved in the mechanism of G protein-coupled receptor agonist- or tumor necrosis factor (TNF)-α-induced cardiomyocyte hypertrophy in cultured rat neonatal cardiomyocytes.6,7 The in vivo role of the NF-κB signaling pathway in the heart has been investigated using mice lacking p50. Ablation of p50 improved both cardiac function and survival after myocardial infarction8,9 and in TNF-α-induced cardiomyopathy.10 In contrast, reovirus-induced apoptosis was significantly enhanced in the hearts of p50-deficient mice.11 Thus, the in vivo role of the NF-κB pathway in the pathogenesis of cardiac remodeling remains controversial. Because IKKβ is both necessary and sufficient for phosphorylation of IκBs in the canonical NF-κB signaling pathway, cardiomyocyte-specific knockdown of IKKβ enables investigation of the precise role of NF-κB in cardiomyocytes. The purpose of the present study was to determine whether the IKKβ/NF-κB pathway plays a protective or harmful role in the response of cardiomyocytes to hemodynamic stress, using cardiac-specific IKKβ-deficient mice.
Materials and Methods
Generation of Cardiac-Specific IKKβ-Deficient Mice
We mated Ikbkβflox/flox mice12 with knock-in mice expressing Cre recombinase under control of the myosin light chain 2V promoter (MLC2V-Cre mice)13 to generate Ikbkβflox/flox;MLC2V-Cre+ mice, which harbored the cardiac-specific IKKβ knockout.
This study was carried out under the supervision of the Animal Research Committee of Osaka University and in accordance with the Guidelines for Animal Experiments of Osaka University and the Japanese Animal Protection and Management Law (No. 25).
Transverse Aortic Constriction, Echocardiography, and Hemodynamic Analysis
We performed transverse aortic constriction (TAC), murine transthoracic echocardiography, and hemodynamic measurements.2 Noninvasive measurements of blood pressure were carried out on mice anesthetized with 2.5% avertin using a BP Monitor for Rats and Mice Model MK-2000 (Muromachi Kikai, Tokyo, Japan) according to the instructions of the manufacturer.
Histological Analysis and Evaluation of Apoptosis
Hematoxylin/eosin or Azan-Mallory staining was performed on paraffin-embedded sections. The cross-sectional areas of cardiomyocytes were determined as previously described.2 To detect apoptotic cells, triple staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), propidium iodide, and anti-α-sarcomeric actin antibody (Sigma) and immunohistochemical staining for activated caspase 3 using anti-activated caspase 3 antibody (Abcam Inc) were performed using paraffin-embedded sections.14 To detect nuclear accumulation of the NF-κB subunit, we performed immunohistochemical staining for p65 on paraffin-embedded sections with anti-p65 antibody (Santa Cruz Biotechnology).
Western Blot Analysis
Protein homogenates were subjected to Western blot analysis using antibodies against IKKα (IMGENEX), IKKβ (Abcam), NEMO and total c-Jun N-terminal kinase (JNK) (Santa Cruz Biotechnology), phospho-JNK (Cell Signaling Technology), and manganese superoxide dismutase (MnSOD).15 Signals of the scanned autoradiographs were quantified by densitometric measurements with the Scion Image software (version 4.02; Scion corp.).
Preparation of Nuclear Extract and the Electrophoretic Mobility Shift Assay
Electrophoretic mobility-shift assay (EMSA) was performed as previously described.7
Total RNA Extraction, RNA Dot Blot Analysis, and Quantitative Real-Time RT-PCR
Isolation of total RNA and quantitative assessment of mRNA expression by dot blot analysis or quantitative RT-PCR were performed.2 PCR primers and probes for TNF-α and interleukin (IL)-2 has been previously described.2 Primers and probes for other targets were obtained from Applied Biosystems. RT-PCR standard curves were constructed using the corresponding cDNA. All data were normalized to GAPDH content and are expressed as fold increase relative to expression in a sham-operated control littermate mouse.
We immunoprecipitated the IKK complex from extracts containing 0.8 mg protein with an anti-NEMO antibody (Santa Cruz Biotechnology). We measured kinase activity using recombinant human IκBα (Cell Science) as the substrate.17
Isolation and Characterization of Adult Mouse Ventricular Myocytes
Mouse adult cardiomyocytes18 were treated with 50 μmol/L isoproterenol (Sigma) alone, 20 μmol/L Mn (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) alone, 20 μmol/L JNK inhibitor II (Calbiochem) alone, 50 μmol/L isoproterenol with 20 μmol/L MnTBAP, or 50 μmol/L isoproterenol with 20 μmol/L JNK inhibitor II. Twenty-four hours after treatment, we determined the viability of cells using trypan blue staining.
Results are shown as means±SEM. Statistical analyses were performed by using Statcel 2 software (OMS Publishing Inc, Tokorozawa, Japan). Comparisons between 2 groups were performed using the Student t test. One-way ANOVAs with Bonferroni’s post hoc test were used for multiple comparisons, and 1-way ANOVAs on ranks were used for multiple comparisons between normalized data. A probability value of <0.05 was considered statistically significant.
Activation of the IKK/NF-κB Pathway in the Pathogenesis of Pressure Overload-Induced Cardiac Remodeling
First, we examined whether the IKK/NF-κB signaling pathway is activated during pressure overload-induced cardiac remodeling. Ten-week-old C57Bl/6J mice were subjected to pressure overload by means of TAC. We performed an in vitro kinase assay to assess the activity of the IKK complex using IκBα as the substrate. The IKK activity in hearts was elevated in response to pressure overload (Figure I, A, in the Online Data Supplement, available at http://circres.ahajournals.org). We subjected nuclear proteins to EMSA for NF-κB DNA-binding activity. We observed increased formation of DNA-protein complexes after TAC (Online Figure I, B). The specificity of NF-κB-binding activity was confirmed by the addition of a 100-fold excess of either unlabeled NF-κB or SP-1 consensus oligonucleotides to the EMSA reaction. Anti-p50 and anti-p65 subunit antibodies supershifted the major NF-κB-binding complex. Furthermore, immunohistochemical analysis for p65 subunit of NF-κB showed the increase in nuclear accumulation of p65 in response to pressure overload (Online Figure I, C). These findings indicate that pressure overload activates the IKK/NF-κB signaling pathway.
Generation of Cardiac-Specific IKKβ-Deficient Mice
To investigate the in vivo role of IKK/NF-κB activation in response to pressure overload, we generated cardiac-specific IKKβ-deficient mice. We crossed Ikbkβ flox/flox mice12 with MLC2V-Cre mice to generate Ikbkβ flox/flox;MLC2V-Cre+ mice (CKO). We used Ikbkβ flox/flox;MLC2V-Cre− littermates as controls (CTRL). Immunoblot analysis of hearts from 10-week-old mice indicated an ≈60% reduction in IKKβ protein level in CKO hearts relative to protein expression in CTRL hearts (Figure 1A). There were no differences in the expression levels of other IKK complex components such as IKKα and NEMO between CKO and CTRL (Figure 1A).
Normal Cardiac Structure and Function in IKKβ-Deficient Mice
The CTRL and CKO mice were born at the expected mendelian ratios (n=76 and 77, respectively). The CKO mice were normal at birth, and their external appearance was indistinguishable from that of their CTRL littermates. The mice grew to adulthood and were fertile. There was no difference in survival at 1 year between CTRL and CKO. Thus, IKKβ is not essential for cardiac development in mice.
The CKO hearts showed no evidence of cardiac morphological defects. Histological examination of the hearts from 10-week-old mice demonstrated no myofibrillar disarray, necrosis, or ventricular fibrosis under basal conditions (data not shown). In addition, heart weight, hemodynamic data, and echocardiographic parameters (Tables 1 and 2⇓) were not significantly different between CKO and CTRL. These findings indicate that CKO had normal global cardiac structure and function.
Development of Congestive Heart Failure in Response to Pressure Overload in IKKβ-Deficient Mice
We subjected 10-week-old CKO and CTRL to pressure overload by means of TAC. First, we examined whether pressure overload-induced IKK/NF-κB activation is efficiently inhibited in CKO hearts. Cytosolic and nuclear proteins were extracted from hearts 2 days after TAC and subjected to either in vitro kinase assay or EMSA (Figure 1B and 1C). TAC induced the activation of IKK and the formation of NF-κB-DNA complexes in CTRL after TAC. NF-κB activation was attenuated in CKO hearts. The specificity of NF-κB-binding activity was confirmed by the competition assay and supershift assay using anti-p50 and anti-p65 subunit antibodies. One week after TAC, we performed echocardiographic analysis (Figure 2A). The diastolic interventricular septum thickness and diastolic left ventricular (LV) posterior wall thickness were not significantly different between CKO and CTRL subjected to TAC (Figure 2B and 2C). One week after sham operation, the end-diastolic LV dimension and LV fractional shortening were not significantly different between CTRL and CKO. However, end-diastolic LV dimension was significantly elevated and LV fractional shortening had significantly reduced in TAC-operated CKO compared with both sham-operated CKO and TAC-operated CTRL (Figure 2D and 2E). Furthermore, the lung/body weight ratio, an index of lung congestion, was significantly elevated in TAC-operated CKO compared with the other groups (Figure 2F). Only 1 of 19 CKO mice died before 1 week after TAC, whereas all CTRL mice (n=15) lived up to 1 week after TAC. These findings indicate that CKO developed congestive heart failure in response to pressure overload.
Enhancement of Pressure Overload-Induced Cardiac Remodeling and Fibrosis in IKKβ-Deficient Mice
Although the ratio of heart weight/body weight was not significantly different between sham-operated groups 1 week after surgery, the ratio was significantly higher in TAC-operated CKO than TAC-operated CTRL (Figure 3A). TAC increased cardiomyocyte cross-sectional area in both CKO and CTRL (Figure 3B). However, cardiomyocyte cross-sectional area was significantly greater in TAC-operated CKO than in TAC-operated CTRL. We evaluated the level of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) mRNA expression by means of dot blot analysis (Figure 3C). ANF and BNP, which are biochemical markers of cardiac remodeling, were not significantly different between sham-operated groups but were significantly higher in TAC-operated CKO than in TAC-operated CTRL. Two days after TAC, when LV function is similar in both groups, the extent of cardiac hypertrophy was not significantly different between CTRL and CKO (data not shown). These findings suggest that enhanced cardiac hypertrophy in CKO 1 week after TAC was secondary to LV dysfunction.
Azan-Mallory staining demonstrated that interstitial and perivascular fibrosis was present in both CTRL and CKO hearts after TAC, but the extent of fibrosis in CKO was much greater than in CTRL (Figure 3D). The induction of both α1 type I and type III collagen mRNA 1 week after TAC was significantly enhanced in CKO relative to expression in CTRL (Figure 3E).
Increased Cardiomyocyte Apoptosis in IKKβ-Deficient Mice
We evaluated cardiomyocyte apoptosis in hearts 1 week after TAC by means of an in situ TUNEL assay and immunohistochemical analysis using an anti-activated caspase 3 antibody. TUNEL-positive cells contained condensed chromatin and were identified as cardiomyocytes by anti-α-sarcomeric actin staining (Figure 4A). The numbers of TUNEL-positive cardiomyocytes in sham-operated CTRL and sham-operated CKO were similar. However, TAC-operated CKO exhibited more TUNEL-positive cardiomyocytes than TAC-operated CTRL. Although the number of activated caspase 3-positive cardiomyocytes 1 week after TAC was not significantly different between sham-operated CTRL and sham-operated CKO, it was significantly higher in TAC-operated CKO compared with TAC-operated CTRL (Figure 4B). These findings suggest that increased apoptosis might be a cause of heart failure in CKO after TAC.
Downregulation of MnSOD Expression in IKKβ-Deficient Mice After TAC
Next, we investigated the molecular mechanism of increased apoptosis after TAC in CKO. We hypothesized that the impairment of NF-κB target gene expression induced by pressure overload contributes to the increased apoptosis in CKO. Thus, we examined the level of candidate NF-κB-target apoptosis-related genes expression 1 week after TAC. Total RNA was isolated from hearts 1 week after TAC and was subjected to either dot blot analysis or quantitative real-time RT-PCR. The mRNA expression levels of antiapoptotic molecules, such as Bfl-1, c-FLIP, Bcl-xl, XIAP TRAF2, c-IAP1, or Gadd45β, were not significantly different between TAC-operated CTRL and TAC-operated CKO (Figure 5A and 5B). The expression of Bnip3, which is a proapoptotic factor that is downregulated during NF-κB activation, was not significantly different between TAC-operated CTRL and TAC-operated CKO (Figure 5B). We also examined the expression levels of NF-κB-driven inflammatory cytokines. No significant differences in the expression level of TNF-α were detected among the 4 groups (Figure 5B). Expression of IL-1β tended to increase in TAC-operated groups. However, there were no significant differences between TAC-operated CKO and TAC-operated CTRL. IL-2 was not detected in the heart (data not shown).2 Finally, we examined the expression of NF-κB-target antioxidative molecules, such as ferritin heavy chain and MnSOD. We did not detect any differences in ferritin heavy chain mRNA expression among groups (data not shown). MnSOD mRNA was significantly downregulated in TAC-operated groups compared with sham-operated groups. However, downregulation was significantly greater in TAC-operated CKO compared with TAC-operated CTRL (Figure 5C). Consistent with the levels of mRNA expression, protein expression was significantly lower in TAC-operated CKO compared with TAC-operated CTRL (Figure 5D). These findings suggest that enhanced downregulation of MnSOD might contribute to increased apoptosis after TAC in CKO.
Enhanced Oxidative Stress and JNK Activation in IKKβ-Deficient Mice After TAC
To investigate the pathophysiological consequences of MnSOD downregulation, we evaluated the extent of oxidative stress after TAC. Immunohistochemical staining of hearts 1 week after TAC with anti-8-hydroxy-deoxyguanosine (8-OHdG) antibody demonstrated an increase in the number of 8-OHdG-positive cardiomyocytes in CKO compared with CTRL (Figure 6A). Because increased oxidative stress reportedly induces the enhancement of JNK activity and cell death in NF-κB-deficient cells19 and because JNK is a well-known proapoptotic factor, we examined the level of JNK phosphorylation in response to pressure overload. Although the phosphorylation level of p46 JNK was not significantly different between TAC-operated CTRL and TAC-operated CKO, the phosphorylation of p54 JNK was significantly higher in TAC-operated CKO compared with TAC-operated CTRL (Figure 6B). These findings suggest that enhanced oxidative stress and JNK activity mediated through downregulation of MnSOD might be involved in the increased apoptosis observed in CKO after TAC.
Attenuation of Isoproterenol-Induced Cardiomyocyte Death by Both a MnSOD Mimetic and a JNK Inhibitor in CKO
To confirm the involvement of MnSOD downregulation in the mechanism of increased cell death, we tested the effect of a MnSOD mimetic, MnTBAP, on isoproterenol-induced cardiomyocyte death. We isolated cardiomyocytes from adult CTRL and CKO and subjected the cells to the following treatments: isoproterenol alone, MnTBAP alone, or isoproterenol with MnTBAP. MnTBAP has no effect on the expression level of MnSOD (data not shown). We calculated the percentage of trypan blue-negative cardiomyocytes, as an index of cell viability. Under normal conditions, cell viability was not significantly different between CTRL and CKO cardiomyocytes (Figure 7A). Treatment with MnTBAP alone did not affect the viability of either group. Treatment with isoproterenol decreased the viability of CKO cardiomyocytes to a greater extent than CTRL cardiomyocytes. MnTBAP significantly improved the viability of CKO cardiomyocytes treated with isoproterenol.
We also examined the effect of JNK inhibition on isoproterenol-induced cardiomyocyte death (Figure 7B). Treatment with a JNK inhibitor II alone did not affect the viability in either group. The JNK inhibitor II significantly improved the viability of isoproterenol-treated CKO cardiomyocytes. Thus, downregulation of MnSOD and enhanced activation of JNK are both involved in the increased apoptosis in CKO mice after TAC.
In response to pressure overload, the hearts activates an adaptive physiological response in the form of cardiac hypertrophy, whereas long-lasting or excessive exposure to mechanical stress results in transition to heart failure.20 In the present study, we found that the ablation of IKKβ in cardiomyocytes increased pressure overload-induced cardiomyocyte apoptosis, leading to cardiac dysfunction and chamber dilation. These findings indicate that the acute activation of NF-κB in a setting of pressure overload has protective properties. A protective role of the IKKβ/NF-κB signaling pathway in cardiomyocytes is inconsistent with some of previous studies of p50-deficient mice. The ablation of p50 improved cardiac function and survival after myocardial infarction.8,9 However, there was paradoxical increase in TNF-α expression in p50-deficient hearts after myocardial infarction, and it is likely that the use of p50-deficient mice to examine the role of NF-κB pathway is problematic. Although there are some alternate complexes of NF-κB, heterodimers of p65 and p50 are the most common activated complexes. However, after prolonged activation of the p65-p50 dimer, NF-κB is converted from the transcriptionally active p65-p50 heterodimer into the transcriptionally inactive p50-p50 homodimer, which acts as a transcriptional repressor.21 This may be a native negative-feedback mechanism to prevent excessive inflammation. Thus, it is possible that lack of p50-p50 homodimer formation in p50-deficient mice resulted in the paradoxical enhancement of NF-κB activity, which conferred the cardioprotective effects. In addition, Frantz et al also reported that the reduction of cardiac ischemia/reperfusion injury in p50-deficient mice was abolished with transplantation of bone marrow cells isolated from wild-type mice, indicating that the primary mechanism of injury is activation of NF-κB in infiltrating leukocytes and not in cardiomyocytes.22 Taken together, these observations suggest that p50-deficient mice may not be the appropriate model to clarify the role of the NF-κB pathway in cardiomyocytes. Thus, we used cardiomyocyte-specific IKKβ-deficient mice in this study. We efficiently inhibited activation of both the cardiac IKK complex and NF-κB in response to pressure overload using this model.
The role of the pathway differs among cell types. Enterocyte-specific knockout of IKKβ prevented a systemic inflammation but resulted in severe apoptotic damage to the reperfused intestinal mucosa.17 In contrast, hepatocyte-specific knockout of IKKβ prevented ischemia/reperfusion-induced necrotic injury in the liver,23 and neuron-specific knockout of IKKβ significantly ischemia-induced apoptosis of neuron.24 These findings indicate that in vivo, NF-κB can have different effects on the mechanism of cell death, depending on the organ or type of stimulus.
Here, we found that the IKKβ/NF-κB pathway is a negative regulator of programmed cell death. NF-κB induces the transcriptional activation of antiapoptotic and antioxidant molecules.25 Quantification of mRNA expression of NF-κB-dependent antiapoptotic genes revealed that MnSOD is responsible for the protective role of NF-κB. MnSOD was severely downregulated in IKKβ-deficient hearts after TAC. Sustained JNK activation, which has been observed in IKKβ- or RelA-deficient cells, promotes apoptosis.19 JNK activation is mediated by the downregulation of MnSOD, reactive oxygen species accumulation, and resultant inactivation of mitogen-activated protein kinase (MAPK) phosphatase.19 We observed enhanced oxidative stress and JNK activation in pressure-overloaded hearts of mice. However, we did not measure MAPK phosphatase activity, because it is technically difficult to estimate its specific activity in hearts. Given that the activity of apoptosis signal-regulating kinase 1, which is an upstream kinase of JNK, was not significantly different between control and IKKβ-deficient hearts (data not shown), it is possible that inactivation of MAPK phosphatase is involved in the enhanced JNK activation observed in IKKβ-deficient hearts. Furthermore, we examined whether IKKβ/NF-κB-MnSOD-JNK linkage is associated with the protective mechanism in cardiomyocytes using cardiomyocytes isolated from adult mice. We found that the IKKβ-deficient cardiomyocytes were more susceptible to isoproterenol-induced death and that the susceptibility was attenuated by treatment with either a MnSOD mimetic or a JNK inhibitor. These results indicate that this pathway is a critical component of the cardiomyocyte defense against acute hemodynamic stress. However, we cannot exclude the possibility that other molecules than MnSOD may contribute to increased apoptosis in CKO. In addition, considering that this pathway functions proapoptotic in other organs, the function of NF-κB pathway may be different in the setting of chronic heart failure.
Although the expression of MnSOD decreased in response to pressure overload, the magnitude of the decrease was enhanced in pressure-overloaded IKKβ-deficient hearts. However, the molecular mechanism of the pressure overload-induced downregulation in IKKβ-deficient mice remains to be elucidated. A series of transcription factors, such as specificity protein-1, activator protein-2, CCAAT/enhancer-binding protein, p53, and NF-κB, reportedly regulate MnSOD gene expression.26–28 Among these factors, activation of NF-κB is essential, but not sufficient, for the induction of MnSOD.26 In contrast, it is well known that p53 functions as a negative regulator of MnSOD gene transcription and that p53 is induced in response to pressure overload in the heart.29 NF-κB has been shown to antagonize p53 transactivation through the induction of p53 proteolysis or sequestration of the p300 and CREB-binding protein coactivators.30,31 Thus, the absence of NF-κB-driven p53 inhibition after TAC might decrease MnSOD in IKKβ-deficient mice.
In conclusion, we demonstrated that IKKβ/NF-κB plays a protective role in the cardiomyocyte response to pressure overload. The IKKβ/NF-κB signaling pathway may constitute a molecular target for the prevention and treatment of heart failure.
We are grateful to M. Karin (University of California at San Diego) for the gift of Ikbkβflox/flox mice and K. Chien (Harvard University) for the gift of MLC2V-Cre mice.
Sources of Funding
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to K.O. and S.H.) and from Takeda Science Foundation (to S.H.).
Original received December 25, 2008; revision received April 28, 2009; accepted May 20, 2009.
Yamaguchi O, Higuchi Y, Hirotani S, Kashiwase K, Nakayama H, Hikoso S, Takeda T, Watanabe T, Asahi M, Taniike M, Matsumura Y, Tsujimoto I, Hongo K, Kusakari Y, Kurihara S, Nishida K, Ichijo H, Hori M, Otsu K. Targeted deletion of apoptosis signal-regulating kinase 1 attenuates left ventricular remodeling. Proc Natl Acad Sci U S A. 2003; 100: 15883–15888.
Hikoso S, Yamaguchi O, Higuchi Y, Hirotani S, Takeda T, Kashiwase K, Watanabe T, Taniike M, Tsujimoto I, Asahi M, Matsumura Y, Nishida K, Nakajima H, Akira S, Hori M, Otsu K. Pressure overload induces cardiac dysfunction and dilation in signal transducer and activator of transcription 6-deficient mice. Circulation. 2004; 110: 2631–2637.
Hayden MS, Ghosh S. Signaling to NF-κB. Genes Dev. 2004; 18: 2195–2224.
Hirotani S, Otsu K, Nishida K, Higuchi Y, Morita T, Nakayama H, Yamaguchi O, Mano T, Matsumura Y, Ueno H, Tada M, Hori M. Involvement of nuclear factor-κB and apoptosis signal-regulating kinase 1 in G-protein-coupled receptor agonist-induced cardiomyocyte hypertrophy. Circulation. 2002; 105: 509–515.
Kawano S, Kubota T, Monden Y, Tsutsumi T, Inoue T, Kawamura N, Tsutsui H, Sunagawa K. Blockade of NF-κB improves cardiac function and survival after myocardial infarction. Am J Physiol Heart Circ Physiol. 2006; 291: H1337–H1344.
Frantz S, Hu K, Bayer B, Gerondakis S, Strotmann J, Adamek A, Ertl G, Bauersachs J. Absence of NF-κB subunit p50 improves heart failure after myocardial infarction. FASEB J. 2006; 20: 1918–1920.
Kawamura N, Kubota T, Kawano S, Monden Y, Feldman AM, Tsutsui H, Takeshita A, Sunagawa K. Blockade of NF-κB improves cardiac function and survival without affecting inflammation in TNF-α-induced cardiomyopathy. Cardiovasc Res. 2005; 66: 520–529.
O'Donnell SM, Hansberger MW, Connolly JL, Chappell JD, Watson MJ, Pierce JM, Wetzel JD, Han W, Barton ES, Forrest JC, Valyi-Nagy T, Yull FE, Blackwell TS, Rottman JN, Sherry B, Dermody TS. Organ-specific roles for transcription factor NF-κB in reovirus-induced apoptosis and disease. J Clin Invest. 2005; 115: 2341–2350.
Li ZW, Omori SA, Labuda T, Karin M, Rickert RC. IKKβ is required for peripheral B cell survival and proliferation. J Immunol. 2003; 170: 4630–4637.
Chen J, Kubalak SW, Minamisawa S, Price RL, Becker KD, Hickey R, Ross J Jr, Chien KR. Selective requirement of myosin light chain 2v in embryonic heart function. J Biol Chem. 1998; 273: 1252–1256.
Hikoso S, Ikeda Y, Yamaguchi O, Takeda T, Higuchi Y, Hirotani S, Kashiwase K, Yamada M, Asahi M, Matsumura Y, Nishida K, Matsuzaki M, Hori M, Otsu K. Progression of heart failure was suppressed by inhibition of apoptosis signal-regulating kinase 1 via transcoronary gene transfer. J Am Coll Cardiol. 2007; 50: 453–462.
Fujii J, Ikeda Y, Watanabe T, Kawasaki Y, Suzuki K, Fujii C, Takahashi M, Taniguchi N. A defect in the mitochondrial import of mutant Mn-superoxide dismutase produced in Sf21 cells. J Biochem. 1998; 124: 340–346.
Deleted in proof.
Luedde T, Assmus U, Wustefeld T, Meyer zu Vilsendorf A, Roskams T, Schmidt-Supprian M, Rajewsky K, Brenner DA, Manns MP, Pasparakis M, Trautwein C. Deletion of IKK2 in hepatocytes does not sensitize these cells to TNF-induced apoptosis but protects from ischemia/reperfusion injury. J Clin Invest. 2005; 115: 849–859.
Jones PL, Ping D, Boss JM. Tumor necrosis factor α and interleukin-1β regulate the murine manganese superoxide dismutase gene through a complex intronic enhancer involving C/EBP-β and NF-κB. Mol Cell Biol. 1997; 17: 6970–6981.
Dhar SK, Xu Y, Chen Y, St Clair DK. Specificity protein 1-dependent p53-mediated suppression of human manganese superoxide dismutase gene expression. J Biol Chem. 2006; 281: 21698–21709.
Sano M, Minamino T, Toko H, Miyauchi H, Orimo M, Qin Y, Akazawa H, Tateno K, Kayama Y, Harada M, Shimizu I, Asahara T, Hamada H, Tomita S, Molkentin JD, Zou Y, Komuro I. p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload. Nature. 2007; 446: 444–448.
Webster GA, Perkins ND. Transcriptional cross talk between NF-κB and p53. Mol Cell Biol. 1999; 19: 3485–3495.