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Am J Physiol Heart Circ Physiol 292: H530-H538, 2007. First published August 18, 2006; doi:10.1152/ajpheart.00549.2006
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Inhibition of NF-{kappa}B improves left ventricular remodeling and cardiac dysfunction after myocardial infarction

Yasuyuki Onai,1 Jun-ichi Suzuki,1 Yasuhiro Maejima,1 Go Haraguchi,1 Susumu Muto,2 Akiko Itai,2 and Mitsuaki Isobe1

1Department of Cardiovascular Medicine, Tokyo Medical and Dental University; and 2Institute of Medicinal Molecular Design, Tokyo, Japan

Submitted 29 May 2006 ; accepted in final form 12 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Several studies have demonstrated that NF-{kappa}B is substantially involved in the progression of cardiac remodeling; however, it remains uncertain whether the continuous inhibition of NF-{kappa}B is effective for the prevention of myocardial remodeling. Myocardial infarction (MI) was produced by ligation of the left anterior coronary artery of rats. IMD-0354 (10 mg/kg per day), a novel phosphorylation inhibitor of I{kappa}B that acts via inhibition of IKK-beta, was injected intraperitoneally starting 24 h after induction of MI for 28 days. After 28 days, the IMD-0354-treated group showed significantly improved survival rate compared with that of the vehicle-treated group (P < 0.05). Although infarct size was similar in both groups, improved left ventricular (LV) remodeling and diastolic dysfunction, as indicated by smaller LV cavity (LV end-diastolic area: vehicle, 74.13 ± 3.57 mm2; IMD-0354, 55.00 ± 3.73 mm2; P < 0.05), smaller peak velocity of early-to-late filling wave (E/A) ratio (vehicle, 3.87 ± 0.26; IMD-0354, 2.61 ± 0.24; P < 0.05), and lower plasma brain natriuretic peptide level (vehicle, 167.63 ± 14.87 pg/ml; IMD-0354, 110.75 ± 6.41 pg/ml; P < 0.05), were observed in the IMD-0354-treated group. Moreover, fibrosis, accumulation of macrophages, and expression of several factors (transforming growth factor-beta1, monocyte chemoattractant protein-1, matrix metalloproteinase-9 and -2) in the noninfarcted myocardium was remarkably inhibited by IMD-0354. In conclusion, inhibition of NF-{kappa}B activation may reduce the proinflammatory reactions and modulate the extracellular matrix and provide an effective approach to prevent adverse cardiac remodeling after MI.

experimental heart failure; nuclear factor-{kappa}b; matrix metalloproteinase; echocardiography

MYOCARDIAL INFARCTION (MI) frequently leads to left ventricular (LV) dilatation and associated interstitial fibrosis in the noninfarcted myocardium. This adverse LV remodeling causes depressed cardiac performance and is an independent determinant of morbidity and mortality after MI (20). One of the recent conceptual advances in our understanding of the pathogenesis of LV remodeling is that this process is driven by the overexpression of various factors, including proinflammatory cytokines, angiotensin II, and norepinephrine, which have direct pathophysiological effects on cardiac myocytes, noncardiac myocytes, and the extracellular matrix (20). Although the expression of proinflammatory cytokines may be involved in wound healing after MI, it is believed that the overexpression of cytokines damages cardiac tissue and evokes excess deposition of fibrotic components, even in the noninfarcted myocardium.

Several recent studies reported that nuclear factor-{kappa}B (NF-{kappa}B) is involved in the pathogenesis of heart failure (7, 35). Activation of NF-{kappa}B induces activation of genetic programs that lead to transactivation of cytokines, chemokines, and matrix metalloproteinases (MMPs), promoting inflammatory and fibrotic responses that participate in the progression of myocardial remodeling. NF-{kappa}B is a dimer of Rel family members, and the most common active form is composed of p50 or p52 and p65. In resting cells, NF-{kappa}B is inactive and is segregated in the cytoplasm, bound to an inhibitory protein known as the inhibitor of NF-{kappa}B (I{kappa}B). NF-{kappa}B activation requires phosphorylation of I{kappa}B by the I{kappa}B kinase (IKK) complex. Phosphorylated I{kappa}B is then ubiquitinated and degraded by proteasomes. Subsequently, unbound NF-{kappa}B is translocated to the nucleus and binds to the promoter or enhancer of specific genes, including those involved in inflammatory reactions (17). Recent studies have suggested that these inflammatory reactions may cause myocardial damage and fibrosis (6). Since inflammation evokes further activation of inflammatory status, possibly representing a vicious cycle, modulation of NF-{kappa}B might have a potent effect on ablating inflammatory linkage operative in the development of LV remodeling.

Despite several studies that have mentioned the potential role of NF-{kappa}B in the pathogenesis of cardiac remodeling, only a few studies have examined the effect of continuous inhibition of NF-{kappa}B on myocardial remodeling after MI (15, 30), and its mechanism has not been fully elucidated yet. Therefore, we sought to determine whether blocking of NF-{kappa}B by phosphorylation inhibitor of I{kappa}B, IMD-0354 (IMD), ameliorates progressive LV remodeling after experimental MI.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Surgical procedure. Eight- to ten-week-old male Sprague-Dawley rats (250–300 g) were anesthetized with pentobarbital sodium (40–60 mg/kg ip) and intubated orally with a polyethylene tube for artificial respiration (model SN-480-7, Shinano, Tokyo, Japan). After a thoracotomy was performed at the fourth intercostal space, the pericardium was gently removed to expose the heart. Myocardial ischemia was produced by ligation of the left anterior descending coronary artery (LAD). The chest was then closed, and the animals were allowed to recover in a warm, clean cage. Rats that died within 24 h of the operation were excluded from this study. All animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). The protocol was approved by the Animal Research Committee of the Tokyo Medical and Dental University.

Reagents. IMD-0354 [N-(3,5-bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide; IMD] was kindly provided by the Institute of Medicinal Molecular Design (Tokyo, Japan). The drug was dissolved in 0.5% carboxymethylcellulose (CMC, Sigma Chemical, Tokyo, Japan) immediately before use. The efficacy and pharmacological mechanism of IMD have been determined in previous studies (14, 23, 29). Moreover, drug safety of IMD up to 28 days also has been presented in a previous study (23). Vehicle (0.5% CMC) was used as a control.

Treatment protocol. Animals were assigned randomly to one of three treatment groups 24 h after operation, as follows: IMD-0354 injection (10 mg/kg ip) daily for 28 days (MI + IMD group, n = 20); vehicle injection (intraperitoneally) daily for 28 days (MI + vehicle group, n = 20); and sham-operation (thoracotomy with LAD isolation but without ligation, n = 9).

Measurement of heart size and cardiac function. Twenty-eight days after induction of MI, transthoracic echocardiography was carried out with an ultrasound machine (Sonos 5500; Philips, Andover, MA) and a 12-MHz annular array transducer under light pentobarbital sodium anesthesia. Hearts were imaged with the two-dimensional mode in short-axis views at the level of the papillary muscles to measure the LV end-diastolic dimension (LVEDD), LV end-systolic dimension, LV end-diastolic area (LVEDA), and ratio of fractional shortening (in %). Ejection fraction (in %) was calculated by the area-length method. As an index of LV diastolic function, the ratio of the peak velocity of early (E) to late (A) filling waves and deceleration time (DcT) of E waves were determined by Doppler traces of the mitral flow from the apical four-chamber view as described previously (26). Each value was presented as the average of measurements of three consecutive beats obtained by blinded observers.

Measurement of infarct size and histological analysis. After experiments, rats were killed with an overdose intraperitoneal injection of pentobarbital sodium. The right and left ventricles of harvested hearts were dissected and weighed. Five-micrometer-thick sections were obtained from the LV and were stained with Mallory-Azan stain as described previously (38), and the stained area was detected as infarcted area. Infarct size was then calculated as the total infarct circumference divided by total LV circumference (n = 8, each group). The area of myocardial fibrosis in the noninfarcted myocardium (middle portion of LV septic wall) (n = 6; each group) was measured and corrected for the area of the microscopic field in a blinded manner at a magnification of x200 with Scion Image beta4.0.2 software (Meyer Instruments, Houston, TX). Three fields of each section were averaged and used for the fibrosis area. Perivascular fibrosis was excluded from the fibrosis area.

Immunohistochemistry. Hearts were cut at the level of the papillary muscles and frozen in optimum cutting temperature compound (Sakura Finetek, Tokyo, Japan). Each section (5 µm) was incubated with primary antibodies, ED1 (Serotec, Oxford, UK) and {alpha}-smooth muscle actin (Sigma Chemical), and then with Histofine Simple Stain Rat (Nichirei, Tokyo, Japan), followed by reaction with AEC matrix solution (Nichirei). Immunostained type- and class-matched nonimmune IgGs were used as negative controls.

Preparation of protein extracts. Heart sections obtained from the LV septum were homogenized in extraction buffer containing 50 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 2 mM EGTA, 10 mM EDTA, 100 mM NaF, 1 mM Na4P2O7, 2 mM Na3VO4, 100 µg/ml PMSF, and protease inhibitor. After centrifugation, the second supernatant layer was collected for the analysis of heart protein. The separation of cytosolic and nuclear protein was carried out according to the previous study (10, 23). Protein concentrations were measured with a Bio-Rad protein assay kit (Bio-Rad, Milan, Italy) to equalize the protein concentrations of all samples.

Western blot analysis and ELISA. Equal amounts of proteins were loaded and separated by SDS-PAGE, transferred to nitrocellulose membrane, and incubated with monoclonal antibody to I{kappa}B-{alpha} (Cell Signaling, Beverly, MA), phospho-I{kappa}B-{alpha} (Cell Signaling), NF-{kappa}B p65 (Santa Cruz Biotechnology, Santa Cruz, CA), lamin A/C (Chemicon International, Temecula, CA), transforming growth factor-beta1 (TGF-beta1; Santa Cruz Biotechnology), or monocyte chemoattractant protein-1 (MCP-1; Santa Cruz Biotechnology). The membranes were incubated with secondary antibody (Amersham Biosciences, Piscataway, NJ) and developed with enhanced chemiluminescence reagent (Amersham Biosciences). Enhanced chemiluminescence was detected with an LAS-1000 image analyzer system (Fujifilm, Tokyo, Japan). The plasma concentration of brain natriuretic peptide (BNP) at 28 days after induction of MI was determined by ELISA (Angiopharm, O'Fallon, MO) according to the manufacturer's instructions.

Isolation and treatment of cardiac fibroblasts. Neonatal rat cardiac fibroblasts were isolated from the hearts of 1- or 2-day-old Wistar rats as described previously (3). Second-to-fourth passage cells were used for all experiments to exclude contamination by cardiomyocytes. After the starvation period of 48 h, cells were treated with IL-1beta (4 ng/ml) or TNF-{alpha} (100 ng/ml) in the presence or absence of IMD (0.1 or 1.0 µM) for 48 h. In vitro dosage of IMD was standardized to the in vivo blood concentration curve (data not shown).

Gelatin zymography. For in vitro sample preparation, conditioned media of fibroblasts were collected, centrifuged to remove cells and debris, and concentrated. For in vivo sample preparation, the LV septum was obtained at 7 days after induction of MI, washed with cold PBS, and snap frozen in liquid nitrogen. For extraction, tissues were minced into 1-mm3 pieces and incubated with 0.5% Triton X-100 in PBS containing 0.01% sodium azide. The samples were then centrifuged, and the supernatants were collected. The MMP activity of in vitro samples (500 ng protein) and in vivo samples (100 µg protein) was measured by in-gel zymography with gelatin (1 mg/ml, type A from porcine skin; Sigma Chemical) as the substrate, as described previously (27). Enzyme activity attributed to MMP-9 and MMP-2 was visualized as clear bands against a blue background. Recombinant human MMP-9 and MMP-2 (Biomol, Plymouth Meeting, PA) were included in the gels as standards. Each MMP activity was quantified with an imaging densitometer.

Statistical analysis. Results are presented as means ± SE. All data were analyzed by analysis of variance followed by Scheffé's test for multiple comparisons. Mann-Whitney U-test was used to compare survival rates between two groups. A P value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Mortality. The survival rate within 24 h of ligation of the coronary artery was ~40%. Surviving rats were assigned to the MI + vehicle group (n = 20) or the MI + IMD group (n = 20). The survival rate up to 28 days was significantly greater in the MI + IMD group than in the MI + vehicle group (P < 0.05). The numbers of rats that survived up to 28 days were 8 rats in the vehicle-treated group and 13 rats in the IMD-treated group (Fig. 1). There were no differences in infarct size; the MI + vehicle group showed an average infarct size of 50.83 ± 2.26%, whereas the MI + IMD group showed an average infarct size of 50.08 ± 1.72% (P = 0.99).


Figure 1
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  		Fig. 1. Survival rates of rats with myocardial infarction (MI). Percentages of rats surviving MI + vehicle (n = 20) and of rats surviving MI + IMD-0354 (MI + IMD; n = 20) were plotted. At 28 days after induction of MI, the survival rate of the MI + IMD group was significantly better than that of the MI + vehicle group. All sham-operated rats survived during the follow-up period of 28 days (not shown).

 
Heart failure after induction of MI. Twenty-eight days after induction of MI, the right ventricle weight-to-body weight ratio and lung weight-to-body weight ratio were significantly greater in the MI + vehicle group than in the sham-operated group. Treatment with IMD decreased both of these ratios and also plasma BNP remarkably in comparison with those in the MI + vehicle group, indicating that development of heart failure was inhibited by treatment with IMD. There was no significant difference in the LV weight-to-body weight ratio in any group 28 days after induction of MI (Table 1).


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  		Table 1. Infarct size, organ weights, plasma BNP, and echocardiographic assessment

 
IMD prevents activation of NF-{kappa}B in vivo. NF-{kappa}B activity in noninfarcted myocardium was increased in MI + vehicle rats on day 7, consistent with a previous report that showed that the increased activity of NF-{kappa}B in the noninfarcted region reached the peak value at day 7 (37). The level of cytosolic phospho-I{kappa}B{alpha} in the noninfarcted region was decreased, together with prevention of nuclear translocation of p65 NF-{kappa}B by treatment with IMD, as shown by Western blot analysis (Fig. 2).


Figure 2
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  		Fig. 2. Representative Western blot results for cytosolic I{kappa}B{alpha}, phospho-I{kappa}B{alpha}, nuclear p65 NF-{kappa}B, and lamin A/C (A). At 7 days after induction of MI, phosphorylation of cytosolic I{kappa}B and nuclear translocation of p65 NF-{kappa}B were increased in the myocardium of MI + vehicle rats, and these increases were suppressed by treatment with IMD-0354. Ratio of cytosolic phospho-I{kappa}B{alpha} to I{kappa}B{alpha} (B) and percent changes of nuclear p65 NF-{kappa}B (C) were quantified by using an imaging densitometer. Values are means ± SE of 4 experiments. *P < 0.05.

 
Echocardiography. After 28 days, the MI + IMD group showed a significantly smaller increase in LVEDD and LVEDA compared with those in the MI + vehicle group. Although LV systolic function, as indicated by ejection fraction and fractional shortening, was not markedly different between the two groups, significant improvement of diastolic LV function as determined by pulse-wave Doppler recordings of mitral inflow was observed in the MI + IMD group in comparison with the MI + vehicle group. Increased early filling velocity (E), decreased late filling velocity (A), and shortening of DcT of E waves, which are characteristics of failing hearts with elevated LV filling pressure, were observed in the MI + vehicle group. However, the E/A ratio was significantly lower in the MI + IMD group, and DcT of E waves was also improved in the MI + IMD group in comparison with those of the MI + vehicle group with statistical significances. (Fig. 3 and Tables 1 and 2).


Figure 3
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  		Fig. 3. Representative M-mode and short-axis echocardiograms for the MI + vehicle group (A and C; n = 8) and the MI + IMD group (B and D; n = 8) 28 days after induction of MI. LVEDD, left ventricular (LV) end-diastolic dimension. Transmitral flow velocity was obtained from sham (E), MI + vehicle (F), and MI + IMD (G) groups. E, early mitral diastolic wave; A, late mitral diastolic wave.

 

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  		Table 2. Doppler echocardiographic assessment

 
Cardiac fibrosis. Reactive fibrosis in the noninfarcted region adversely affects myocardial stiffness, leading to cardiac dysfunction (33). We examined myocardial fibrosis 28 days after induction of MI. Quantitative histological analysis of Mallory-Azan-stained sections showed a significantly lower extent of interstitial fibrosis in the noninfarcted region of the MI + IMD group compared with that of the MI + vehicle group, whereas very little fibrotic change was detected in hearts of the sham-operated group (Fig. 4).


Figure 4
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  		Fig. 4. Representative photomicrographs of Mallory-Azan-stained LV sections of the noninfarcted region from the sham (A; n = 4), MI + vehicle (B; n = 6), and MI + IMD (C; n = 6) groups at 28 days after induction of MI. Area of cardiac fibrosis in the noninfarcted LV region is presented as percentage of the field (D). *P < 0.05.

 
Inflammatory reactions. We observed no evidence of inflammatory changes in hearts of the sham-operated group (data not shown). Enhanced infiltration of macrophages and myofibroblasts, identified by immunohistochemical staining for ED1 and {alpha}-smooth muscle actin (32), respectively, was observed in myocardium of the MI + vehicle group. These cell types accumulated predominantly in the infarcted area and the border zone; however, numerous infiltrating cells were identified even in the noninfarcted region. Quantification of ED1-positive macrophages and {alpha}-smooth muscle actin-positive myofibroblasts in the noninfarcted regions showed that the numbers of immunopositive cells per section were significantly less in the MI + IMD group than in the MI + vehicle group (Fig. 5). We also examined the protein expression of MCP-1 and TGF-beta1 in the noninfarcted myocardium by Western blot analysis. Cardiac MCP-1 and TGF-beta1 protein levels were significantly increased in the MI + vehicle group compared with the sham-operated group 7 days after induction of MI. This result was consistent with previous reports that showed that both MCP-1 and TGF-beta1 in the infarcted heart reached peaks at day 7 (18, 19). In this study, the increased expression of MCP-1 and TGF-beta1 was significantly reduced in the MI + IMD group compared with the MI + vehicle group (Fig. 6).


Figure 5
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  		Fig. 5. Representative images of immunostaining for ED1 and {alpha}-smooth muscle actin in the sham-operated (A and D; n = 4), MI + vehicle (B and E; n = 6), and MI + IMD (C and F; n = 6) groups. ED1 staining is shown in red, and {alpha}-smooth muscle actin staining is shown in green (FITC). Numbers of infiltrated macrophages (G) and myofibroblasts (H) in sections of the noninfarcted LV myocardium at 7 days after induction of MI. *P < 0.05, **P < 0.01.

 

Figure 6
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  		Fig. 6. Representative Western blot results for transforming growth factor-beta1 (TGF-beta1) and monocyte chemoattractant protein-1 (MCP-1) in the noninfarcted myocardium at 7 days after induction of MI.

 
MMP activity. The gelatinase activity of MMP-9 (95/88 kDa) and MMP-2 (72/66 kDa) in the noninfarcted myocardium at 7 days after induction of MI was significantly increased in the MI + vehicle group compared with that in the sham-operated group, as shown by in-gel gelatin zymography. This increased activity was significantly reduced in the MI + IMD group compared with that in the MI + vehicle group (Fig. 7). In cultured cardiac fibroblasts, IL-1beta-induced MMP-9 activity was almost completely inhibited by IMD, even at the concentration of 0.1 µM. Furthermore, IL-1beta- and TNF-{alpha}-induced MMP-2 activity was attenuated by IMD in a dose-dependent manner (Fig. 8).


Figure 7
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  		Fig. 7. Representative images of in-gel gelatin zymography for matrix metalloproteinase-9 (MMP-9) and MMP-2 in the noninfarcted myocardium at 7 days after induction of MI (A). Recombinant human MMP-9 (95/88 kDa) and MMP-2 (72/66 kDa) were used as positive controls. Relative levels of MMP-9 and MMP-2 activity are expressed as fold increases relative to values in the sham-operated group (B and C). *P < 0.05.

 

Figure 8
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  		Fig. 8. Representative images of in-gel gelatin zymography for MMP-9 and MMP-2 in conditioned media of cultured cardiac fibroblasts stimulated with IL-1beta (A; 4 ng/ml) or TNF-{alpha} (B; 100 ng/ml) in the presence or absence of IMD (0.1 or 1.0 µM). Recombinant human MMP-9 (95/88 kDa) and MMP-2 (72/66 kDa) were used as positive controls (left lanes).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The novel findings of the present study are that 1) blockade of NF-{kappa}B activity with IMD significantly improved mortality and heart failure rates through the suppression of LV geometrical remodeling; 2) IMD reduced accumulation of inflammatory cells in the infarcted heart and suppressed expression of proinflammatory cytokine and chemokine, even in their peak period, contributing to the reduction of myocardial fibrosis; and 3) decrease of MMP activity both in vivo and in vitro may be involved in the beneficial effects of IMD in improving myocardial remodeling.

LV remodeling and NF-{kappa}B inhibition. LV cavity dilatation is one of the compensatory reactions of the failing heart; however, excess dilatation evokes LV systolic and diastolic dysfunction that leads to heart failure. Prevention of unfavorable LV remodeling is important for improvement of morbidity and mortality rates after MI. We created a rat model of large MI that exhibits marked LV dilatation and LV dysfunction, causing heart failure and high mortality rates up to 28 days after induction of MI. Although infarct size in LV was similar in both the MI + vehicle group and the MI + IMD group, LV geometric, structural, and functional remodeling was improved in the MI + IMD group, resulting in lower mortality rates and less heart failure. LV remodeling after MI is produced by activation of multiple factors, including changes in pressure profiles, proinflammatory reactions, and activity of several neurohumoral agents. Recent studies have suggested that an inflammatory response participates in the development of heart failure, and NF-{kappa}B may also be of importance in this respect (7, 35). Actually, it has also been suggested that well-established therapies for the prevention of LV remodeling by angiotensin blockade work, at least in part, through inhibition of increased NF-{kappa}B DNA-binding activity (37). Thus inhibition of the NF-{kappa}B pathway may contribute to improving adverse LV remodeling through the regulation of several factors that lead to an excess adaptation to MI. In the NF-{kappa}B activation pathway, phosphorylation of I{kappa}B by IKK is essential (16). The mechanism of IMD action was presented in this study and previous studies (14, 23, 29), which showed that phosphorylation of I{kappa}B in the cytosol is blocked by IMD via suppression of IKK-beta, resulting in inhibition of nuclear translocation of p65 NF-{kappa}B both in vitro and in vivo. Our findings suggest that direct inhibition of I{kappa}B degradation by IMD may be a useful and potent way to prevent the progression of maladaptive geometrical remodeling after large MI.

Diastolic dysfunction and NF-{kappa}B inhibition. Doppler echocardiography is currently the primary technique for assessing LV diastolic function. Increased E-wave velocity and decreased peak A-wave velocity (or absent A wave), resulting in greater E/A ratios, and decrease of DcT of E waves were observed in our MI rats, and these flow patterns were similar to transmitral flow profiles observed in patients with heart failure with restrictive patterns (8, 21) and in an experimental large MI model in rats (26). In this study, treatment with IMD significantly decreased the E/A ratio and improved the DcT, indicating amelioration of diastolic dysfunction. Improvement in the diastolic filling pattern is caused by preload and/or afterload reduction, improvement in LV relaxation, or decrease in passive elastic properties. Although our data did not directly determine which factor effected improvement of diastolic function in the MI + IMD group, it is possible that the reduction of myocardial fibrosis in the noninfarcted regions contributed to the improvement in LV relaxation and/or elastic properties. Because reactive fibrosis in the noninfarcted regions adversely affects myocardial stiffness, the antifibrotic effect of IMD may prevent LV diastolic dysfunction.

LV remodeling and cytokines. TGF-beta is a locally generated cytokine that has been implicated as a major stimulator of tissue fibroinflammatory changes (1). TGF-beta has a predominant influence on fibroblast proliferation and extracellular matrix production, particularly collagen and fibronectin. TGF-beta also modulates the phenotypic conversion of fibroblasts to myofibroblasts that express {alpha}-smooth muscle actin (28). Myofibroblasts play important roles in a wide range of pathological conditions associated with fibrosis and organ remodeling by producing extracellular components and profibrotic mediators, including TGF-beta (4). In this study, IMD decreased the expression of TGF-beta1 in the heart after MI. Because there are no NF-{kappa}B-binding sites in the promoter region of the TGF-beta1 gene, IMD may contribute indirectly to the expression of TGF-beta1 via reduction of fibroinflammatory reactions, such as infiltration of macrophages and activated myofibroblasts, which are the main sources of TGF-beta. We also examined the expression of MCP-1, a chemotactic factor the expression of which is regulated by NF-{kappa}B (31). There is an evidence that anti-MCP-1 gene therapy can prevent LV remodeling after MI via attenuation of macrophage recruitment and interstitial fibrosis in mice (12). In LV remodeling, macrophages are recruited by MCP-1 and are the main source of inflammatory cytokines (13) and MMPs (2). Thus, MCP-1 plays essential roles in the recruitment of inflammatory cells to promote LV remodeling. Taken together, inhibition of NF-{kappa}B activation to suppress TGF-beta1 and MCP-1 expression may prevent harmful inflammatory conditions that lead to myocardial damage and fibrosis.

LV remodeling and MMPs. Increased expression of certain MMP species has been identified during the development of heart failure. In this study, we showed increased induction of gelatinase activity (i.e., of MMP-9 and MMP-2) in the noninfarcted myocardium 7 days after induction of MI. Recent studies using transgenic mouse models of MMP deletion and MMP inhibitor have provided further insight into the involvement of particular MMP species in LV remodeling after MI (5, 11, 25). These data suggest the importance of gelatinases in the development of LV remodeling. In this study, IMD reduced the increased activity of MMP-9 and MMP-2 that accompanied the inhibition of progressive LV enlargement. Furthermore, we showed that IMD could inhibit the activity of MMP-9 and MMP-2 in cultured cardiac fibroblasts under conditions of cytokine stimulation. Interestingly, MMP-9 was activated by IL-1beta, whereas MMP-2 was activated by both IL-1beta and TNF-{alpha}. This differential regulation of MMP-9 and MMP-2 activation is consistent with a previous report (36). Because IL-1beta and TNF-{alpha} are chronically elevated in myocardium remote from the area of infarct (24) and play crucial roles in the development of LV enlargement, it is noteworthy that IMD decreased the activity of MMPs both in vivo and in vitro under stimulation by these cytokines. Induction or stimulation of MMPs at the transcriptional level is mediated by a variety of inflammatory cytokines, hormones, and growth factors, many of which are regulated by NF-{kappa}B. In fact, MMP-9 has a NF-{kappa}B-binding site in its promoter region (34). Although there is no consensus with regard to the NF-{kappa}B binding site in the MMP-2 promoter, it is possible that NF-{kappa}B modulates MMP-2 activation directly via interaction with other transcriptional factors (36) or indirectly by inducing increased expression of membrane-type 1 MMP (9, 22, 36). Thus, inhibition of NF-{kappa}B by IMD may modulate the activity of MMPs directly and/or indirectly, resulting in improvement of unfavorable LV remodeling, particularly LV enlargement.

Study limitations. Several limitations of the present study should be mentioned. First, we cannot exclude the possibility that the administration of IMD in advance or during the hyperacute period of MI may cause myocardial rupture or progression of LV remodeling via retardation of wound healing. Although there were no unfavorable effects of IMD in the present study, additional experiments with various treatment timings should be examined. Second, the effect of continuous administration and various dosages of IMD should be analyzed to determine the most appropriate timing and dosage of IMD for treatment of cardiac remodeling. Third, though we conclude that improvement of diastolic function is one of the predominant effects of IMD, echocardiographical measurement alone is not adequate to assess diastolic dysfunction accurately. Invasive measurement of diastolic properties would need to be elucidated in further studies.

Clinical implications and conclusion. Because numerous factors are involved in the pathogenesis of adverse cardiac remodeling, it may be impossible to control all the factors. However, because inflammatory reactions, including those of cytokines, chemokines, and MMPs, may play important roles in the progression of cardiac remodeling, blockade of NF-{kappa}B activation is a reasonable therapeutic approach. After MI, proinflammatory factors produced from myocardial tissue, including cardiac myocytes and fibroblasts, may evoke secondary inflammation and amplify and sustain the proinflammatory response to aggravate cardiac damage and fibrosis. Therefore, inhibition of I{kappa}B phosphorylation to block NF-{kappa}B activation has great potential for ablating the vicious feedback loop of inflammation in the myocardium after MI and is useful for preventing the maladaptive LV remodeling. The present study suggests that inhibition of NF-{kappa}B may provide an effective therapy for patients with MI to prevent heart failure.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a grant from the Organization of Pharmaceutical Safety and Research, Grants-in-aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology.


   ACKNOWLEDGMENTS
 
The authors thank Noriko Tamura and Yasuko Matsuda for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Isobe, Dept. of Cardiovascular Medicine, Tokyo Medical and Dental Univ., 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan (e-mail: isobemi.cvm{at}tmd.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Border WA, Noble NA. Transforming growth factor beta in tissue fibrosis. N Engl J Med 331: 1286–1292, 1994.[Free Full Text]
  2. Cipollone F, Prontera C, Pini B, Marini M, Fazia M, De Cesare D, Iezzi A, Ucchino S, Boccoli G, Saba V, Chiarelli F, Cuccurullo F, Mezzetti A. Overexpression of functionally coupled cyclooxygenase-2 and prostaglandin E synthase in symptomatic atherosclerotic plaques as a basis of prostaglandin E2-dependent plaque instability. Circulation 104: 921–927, 2001.
  3. Cowling RT, Gurantz D, Peng J, Dillmann WH, Greenberg BH. Transcription factor NF-kappa B is necessary for up-regulation of type 1 angiotensin II receptor mRNA in rat cardiac fibroblasts treated with tumor necrosis factor-alpha or interleukin-1 beta. J Biol Chem 277: 5719–5724, 2002.[Abstract/Free Full Text]
  4. Desmouliere A, Geinoz A, Gabbiani F, Gabbiani G. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 122: 103–111, 1993.[Abstract/Free Full Text]
  5. Ducharme A, Frantz S, Aikawa M, Rabkin E, Lindsey M, Rohde LE, Schoen FJ, Kelly RA, Werb Z, Libby P, Lee RT. Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest 106: 55–62, 2000.[ISI][Medline]
  6. Frangogiannis NG, Smith CW, Entman ML. The inflammatory response in myocardial infarction. Cardiovasc Res 53: 31–47, 2002.[Abstract/Free Full Text]
  7. Frantz S, Fraccarollo D, Wagner H, Behr TM, Jung P, Angermann CE, Ertl G, Bauersachs J. Sustained activation of nuclear factor kappa B and activator protein 1 in chronic heart failure. Cardiovasc Res 57: 749–756, 2003.[Abstract/Free Full Text]
  8. Giannuzzi P, Imparato A, Temporelli PL, de Vito F, Silva PL, Scapellato F, Giordano A. Doppler-derived mitral deceleration time of early filling as a strong predictor of pulmonary capillary wedge pressure in postinfarction patients with left ventricular systolic dysfunction. J Am Coll Cardiol 23: 1630–1637, 1994.[Abstract]
  9. Han YP, Tuan TL, Wu H, Hughes M, Garner WL. TNF-alpha stimulates activation of pro-MMP2 in human skin through NF-{kappa}B mediated induction of MT1-MMP. J Cell Sci 114: 131–139, 2001.[Abstract]
  10. Haudek SB, Bryant DD, Giroir BP. Differential regulation of myocardial NF kappa B following acute or chronic TNF-alpha exposure. J Mol Cell Cardiol 33: 1263–1271, 2001.[CrossRef][ISI][Medline]
  11. Hayashidani S, Tsutsui H, Ikeuchi M, Shiomi T, Matsusaka H, Kubota T, Imanaka-Yoshida K, Itoh T, Takeshita A. Targeted deletion of MMP-2 attenuates early LV rupture and late remodeling after experimental myocardial infarction. Am J Physiol Heart Circ Physiol 285: H1229–H1235, 2003.[Abstract/Free Full Text]
  12. Hayashidani S, Tsutsui H, Shiomi T, Ikeuchi M, Matsusaka H, Suematsu N, Wen J, Egashira K, Takeshita A. Anti-monocyte chemoattractant protein-1 gene therapy attenuates left ventricular remodeling and failure after experimental myocardial infarction. Circulation 108: 2134–2140, 2003.
  13. Herskowitz A, Choi S, Ansari AA, Wesselingh S. Cytokine mRNA expression in postischemic/reperfused myocardium. Am J Pathol 146: 419–428, 1995.[Abstract]
  14. Kamon J, Yamauchi T, Muto S, Takekawa S, Ito Y, Hada Y, Ogawa W, Itai A, Kasuga M, Tobe K, Kadowaki T. A novel IKKbeta inhibitor stimulates adiponectin levels and ameliorates obesity-linked insulin resistance. Biochem Biophys Res Commun 323: 242–248, 2004.[CrossRef][ISI][Medline]
  15. Kawano S, Kubota T, Monden Y, Tsutsumi T, Inoue T, Kawamura N, Tsutsui H, Sunagawa K. Blockade of NF-{kappa}B improves cardiac function and survival after myocardial infarction. Am J Physiol Heart Circ Physiol 291: H1337–H1344, 2006.[Abstract/Free Full Text]
  16. Li Q, Estepa G, Memet S, Israel A, Verma IM. Complete lack of NF-kappaB activity in IKK1 and IKK2 double-deficient mice: additional defect in neurulation. Genes Dev 14: 1729–1733, 2000.[Abstract/Free Full Text]
  17. Li Q, Verma IM. NF-kappaB regulation in the immune system. Nat Rev Immunol 2: 725–734, 2002.[CrossRef][ISI][Medline]
  18. Lu L, Chen SS, Zhang JQ, Ramires FJ, Sun Y. Activation of nuclear factor-kappaB and its proinflammatory mediator cascade in the infarcted rat heart. Biochem Biophys Res Commun 321: 879–885, 2004.[CrossRef][ISI][Medline]
  19. Lu L, Zhang JQ, Ramires FJ, Sun Y. Molecular and cellular events at the site of myocardial infarction: from the perspective of rebuilding myocardial tissue. Biochem Biophys Res Commun 320: 907–913, 2004.[CrossRef][ISI][Medline]
  20. Mann DL. Mechanisms and models in heart failure: a combinatorial approach. Circulation 100: 999–1008, 1999.
  21. Mulvagh S, Quinones MA, Kleiman NS, Cheirif J, Zoghbi WA. Estimation of left ventricular end-diastolic pressure from Doppler transmitral flow velocity in cardiac patients independent of systolic performance. J Am Coll Cardiol 20: 112–119, 1992.[Abstract]
  22. Murphy G, Stanton H, Cowell S, Butler G, Knauper V, Atkinson S, Gavrilovic J. Mechanisms for pro matrix metalloproteinase activation. APMIS 107: 38–44, 1999.[ISI][Medline]
  23. Onai Y, Suzuki J, Kakuta T, Maejima Y, Haraguchi G, Fukasawa H, Muto S, Itai A, Isobe M. Inhibition of IkappaB phosphorylation in cardiomyocytes attenuates myocardial ischemia/reperfusion injury. Cardiovasc Res 63: 51–59, 2004.[Abstract/Free Full Text]
  24. Ono K, Matsumori A, Shioi T, Furukawa Y, Sasayama S. Cytokine gene expression after myocardial infarction in rat hearts: possible implication in left ventricular remodeling. Circulation 98: 149–156, 1998.
  25. Peterson JT, Hallak H, Johnson L, Li H, O'Brien PM, Sliskovic DR, Bocan TM, Coker ML, Etoh T, Spinale FG. Matrix metalloproteinase inhibition attenuates left ventricular remodeling and dysfunction in a rat model of progressive heart failure. Circulation 103: 2303–2309, 2001.
  26. Prunier F, Gaertner R, Louedec L, Michel JB, Mercadier JJ, Escoubet B. Doppler echocardiographic estimation of left ventricular end-diastolic pressure after MI in rats. Am J Physiol Heart Circ Physiol 283: H346–H352, 2002.[Abstract/Free Full Text]
  27. Romanic AM, Harrison SM, Bao W, Burns-Kurtis CL, Pickering S, Gu J, Grau E, Mao J, Sathe GM, Ohlstein EH, Yue TL. Myocardial protection from ischemia/reperfusion injury by targeted deletion of matrix metalloproteinase-9. Cardiovasc Res 54: 549–558, 2002.[Abstract/Free Full Text]
  28. Sappino AP, Schurch W, Gabbiani G. Differentiation repertoire of fibroblastic cells: expression of cytoskeletal proteins as marker of phenotypic modulations. Lab Invest 63: 144–161, 1990.[ISI][Medline]
  29. Tanaka A, Konno M, Muto S, Kambe N, Morii E, Nakahata T, Itai A, Matsuda H. A novel NF-{kappa}B inhibitor, IMD-0354, suppresses neoplastic proliferation of human mast cells with constitutively activated c-kit receptors. Blood 105: 2324–2331, 2005.[Abstract/Free Full Text]
  30. Trescher K, Bernecker O, Fellner B, Gyongyosi M, Krieger S, Demartin R, Wolner E, Podesser BK. Adenovirus-mediated overexpression of inhibitor kappa B-alpha attenuates postinfarct remodeling in the rat heart. Eur J Cardiothorac Surg 26: 960–967, 2004.[Abstract/Free Full Text]
  31. Ueda A, Okuda K, Ohno S, Shirai A, Igarashi T, Matsunaga K, Fukushima J, Kawamoto S, Ishigatsubo Y, Okubo T. NF-kappa B and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene. J Immunol 153: 2052–2063, 1994.[Abstract]
  32. Wang J, Chen H, Seth A, McCulloch CA. Mechanical force regulation of myofibroblast differentiation in cardiac fibroblasts. Am J Physiol Heart Circ Physiol 285: H1871–H1881, 2003.[Abstract/Free Full Text]
  33. Weber KT, Brilla CG. Pathological hypertrophy and cardiac interstitium. Fibrosis and renin-angiotensin-aldosterone system. Circulation 83: 1849–1865, 1991.
  34. Westermarck J, Kahari VM. Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J 13: 781–792, 1999.[Abstract/Free Full Text]
  35. Wong SC, Fukuchi M, Melnyk P, Rodger I, Giaid A. Induction of cyclooxygenase-2 and activation of nuclear factor-kappaB in myocardium of patients with congestive heart failure. Circulation 98: 100–103, 1998.
  36. Xie Z, Singh M, Singh K. Differential regulation of matrix metalloproteinase-2 and -9 expression and activity in adult rat cardiac fibroblasts in response to interleukin-1beta. J Biol Chem 279: 39513–39519, 2004.[Abstract/Free Full Text]
  37. Yoshiyama M, Omura T, Takeuchi K, Kim S, Shimada K, Yamagishi H, Teragaki M, Akioka K, Iwao H, Yoshikawa J. Angiotensin blockade inhibits increased JNKs, AP-1 and NF- kappa B DNA-binding activities in myocardial infarcted rats. J Mol Cell Cardiol 33: 799–810, 2001.[CrossRef][ISI][Medline]
  38. Yu Y, Ohmori K, Kondo I, Yao L, Noma T, Tsuji T, Mizushige K, Kohno M. Correlation of functional and structural alterations of the coronary arterioles during development of Type II diabetes mellitus in rats. Cardiovasc Res 56: 303–311, 2002.[Abstract/Free Full Text]



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N. C. Moss, W. E. Stansfield, M. S. Willis, R.-H. Tang, and C. H. Selzman
IKKbeta inhibition attenuates myocardial injury and dysfunction following acute ischemia-reperfusion injury
Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2248 - H2253.
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