AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 287: H1712-H1720, 2004. First published May 13, 2004; doi:10.1152/ajpheart.00124.2004
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NF-{kappa}B activation is required for the development of cardiac hypertrophy in vivo

Yuehua Li,1,3 Tuanzhu Ha,1 Xiang Gao,4 Jim Kelley,2 David L. Williams,1 I. William Browder,1 Race L. Kao,1 and Chuanfu Li1,4

Departments of 1Surgery and 2Internal Medicine, East Tennessee State University, Johnson City, Tennessee 37614; and 3Department of Pathophysiology, Nanjing Medical University, and 4Animal Model Research Center, Nanjing University, Nanjing 210093, China

Submitted 12 February 2004 ; accepted in final form 23 April 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, we examined whether NF-{kappa}B activation is required for cardiac hypertrophy in vivo. Cardiac hypertrophy in rats was induced by aortic banding for 1, 3, and 5 days and 1–6 wk, and age-matched sham-operated rats served as controls. In a separate group of rats, an I{kappa}B-{alpha} dominant negative mutant (I{kappa}B-{alpha}M), a superrepressor of NF-{kappa}B activation, or pyrrolidinedithiocarbamate (PDTC), an antioxidant that can inhibit NF-{kappa}B activation, was administered to aortic-banded rats for 3 wk. The heart weight-to-body weight ratio was significantly increased at 5 days after aortic banding, peaked at 4 wk, and remained elevated at 6 wk compared with age-matched sham controls. Atrial natriuretic peptide and brain natriuretic peptide mRNA expressions were significantly increased after 1 wk of aortic banding, reached a maximum between 2 and 3 wk, and remained increased at 6 wk compared with age-matched sham controls. NF-{kappa}B activity was significantly increased at 1 day, reached a peak at 3 wk, and remained elevated at 6 wk, and IKK-{beta} activity was significantly increased at 1 day, peaked at 5 days, and then decreased but remained elevated at 6 wk after aortic banding compared with age-matched sham controls. Inhibiting NF-{kappa}B activation in vivo by cardiac transfection of I{kappa}B-{alpha}M or by PDTC treatment significantly attenuated the development of cardiac hypertrophy in vivo with a concomitant decrease in NF-{kappa}B activity. Our results suggest that NF-{kappa}B activation is required for the development of cardiac hypertrophy in vivo and that NF-{kappa}B could be an important target for inhibiting the development of cardiac hypertrophy in vivo.

myocardium; signal transduction; nuclear factor-{kappa}B

NUCLEAR FACTOR (NF)-{kappa}B is a ubiquitous inducible transcription factor that activates expression of groups of genes, including those that promote immune and inflammatory responses, cell survival and apoptosis, as well as cell growth and cell cycles, in response to a wide array of extracellular factors (3). In the NF-{kappa}B signaling pathways, there are important kinase cascades, including NF-{kappa}B-inducing kinase (NIK) and I{kappa}B kinase complex (IKK-{alpha} and IKK-{beta}). NIK activates IKKs, which subsequently phosphorylate I{kappa}B-{alpha} at NH2-terminal Ser32 and Ser36 and I{kappa}B-{beta} at Ser19 and Ser23 (8, 28, 38). The phosphorylation and degradation of I{kappa}B proteins are key steps in NF-{kappa}B activation, translocation into the nucleus, and stimulation of gene expression (2, 3). NF-{kappa}B can also be activated through a number of other signaling pathways, including protein kinase C, phosphoinositol 3-kinase (PI3K/Akt), calcium/ nuclear factor of activated T cells (NFAT), Ca2+-calmodulin kinase IV, Ras, gp130, and Janus kinase (JAK)/signal transducers and activators of transcription (STAT)-mediated signaling pathways. Interestingly, all these signaling pathways that can stimulate NF-{kappa}B activation have been documented to play an important role in cardiac hypertrophic responses (16, 29, 31, 34, 35). A role of NF-{kappa}B activation in the development of cardiac hypertrophy in vivo, however, has not been elucidated.

Recent data from in vitro studies suggested that NF-{kappa}B activation is involved in the hypertrophic response of cardiomyocytes. For example, activation of NF-{kappa}B is required for hypertrophic growth of cultured primary rat neonatal ventricular cardiomyocytes (33) and for myotrophin-induced cardiac hypertrophy in vitro (11). Cardiac hypertrophic agonists, such as angiotensin II (ANG II) and endothelin-1, stimulate NF-{kappa}B activation (33), whereas amelioration of ANG II-induced cardiac hypertrophy in vitro was positively correlated with decreased NF-{kappa}B activation. A20, a feedback inhibitor of NF-{kappa}B activation (4), attenuated the hypertrophic response of cardiomyocytes in vitro through the inhibition of NF-{kappa}B signaling (6). Because studies using in vitro cultured cardiac myocytes have significant limitations (32), it is important to elucidate the role of NF-{kappa}B activation in the development of cardiac hypertrophy in vivo. We have previously shown that ischemia-reperfusion (I/R) injury activated NF-{kappa}B in the myocardium both in vitro and in vivo (21–24). Because myocardial hypertrophy is an adaptive response to various stimuli, including myocardial I/R injury and pressure overload, it is possible that NF-{kappa}B activation might be involved in the development of cardiac hypertrophy in vivo.

In the present study, we demonstrated that NF-{kappa}B activation is required for the development of cardiac hypertrophy in vivo. We observed that NF-{kappa}B activation is increased in hypertrophic hearts induced by aortic banding in rats and that specific inhibition of NF-{kappa}B activation by adenovirus-mediated gene transfection of an I{kappa}B-{alpha} dominant negative mutant into the myocardium or treatment with an antioxidant, pyrrolidinedithiocarbamate (PDTC), in vivo attenuates the development of cardiac hypertrophy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Aortic banding-induced cardiac hypertrophy. Male Sprague-Dawley rats (225–250 g) were maintained in the Division of Laboratory Animal Resources at the East Tennessee State University (ETSU) in accordance with the guidelines for the "Principles of Laboratory Animal Care" and the Guide for the Care and Use of Laboratory Animals. All aspects of the animal care and experimental protocols were approved by the ETSU Committee on Animal Care. Cardiac hypertrophy was induced by banding of the ascending aorta with an 18-gauge needle as previously described (18). For the age-matched sham operation, the identical procedure was performed except a suture was not tied around the aorta. The hearts were harvested at 1, 3, and 5 days and 1, 2, 3, 4, 5, and 6 wk after surgical operation with 8–10 rats/group. The heart weight-to-body weight ratio (HW/BW) was calculated, and the heart samples were frozen in liquid nitrogen and then stored at –80°C.

Adenovirus-mediated gene transfection into the myocardium in vivo. Double-deleted adenovirus type 5 (Ad5) expressing an I{kappa}B-{alpha} dominant negative mutant (Ad5-I{kappa}B-{alpha}M, a gift kindly provided by Dr. Yibin Wang, University of Maryland School of Medicine) and Ad5-green fluorescent protein (GFP) were transfected into the myocardium in vivo as previously described (12) with modification. Briefly, rats were anesthetized and ventilated with room air using a rodent ventilator. The hearts were exposed through a left thoracotomy in the third intercostal space. After identification of the aorta, a suture (2-0 silk) was drawn under the ascending aorta. A 22-gauge catheter containing 200 µl of Ad5-GFP or Ad5-I{kappa}B-{alpha}M [1 x 1010 plaque-forming units (pfu)/ml] was advanced from the apex of the left ventricle to the aortic root. The aorta was clamped distal to the site of the catheter, and the solution was then injected. After 10 s, the clamp on the aorta was released and the chest was closed after removal of the air. To examine the transfection efficiency, the hearts were harvested and sectioned 3 and 11 days after transfection, and GFP expression was examined with a fluorescence microscope. To examine the expression of transfected I{kappa}B-{alpha}, the cytoplasmic proteins were isolated from the heart tissue 3 days and 3 wk after transfection and subjected to Western blot analysis with a specific I{kappa}B-{alpha} antibody. To determine the effect of transfected I{kappa}B-{alpha}M on the development of cardiac hypertrophy in vivo, Ad5-I{kappa}B-{alpha}M (1 x 1010 pfu/ml) was transfected into the myocardium immediately followed by banding the aorta, and Ad5-GFP served as a control with 8–10 rats/group. Three weeks after aortic banding, the hearts were harvested, HW/BW was calculated, and heart samples were frozen in liquid nitrogen and then stored at –80°C.

Administration of PDTC. PDTC, an antioxidant that has been shown to have an inhibitory effect on NF-{kappa}B activation, was administered intraperitoneally (80 mg·kg–1·day–1) to aortic-banded or sham-operated rats for 3 wk with 8–10 rats/group. This dose of PDTC has been shown to be effective in blunting NF-{kappa}B activation without significant toxicity (data not shown). The animals were killed, and HW/BW was determined. The heart samples were frozen in liquid nitrogen and then stored at –80°C.

Electrophoretic mobility shift assay. Nuclear proteins were isolated from heart samples, and NF-{kappa}B binding activity was examined by electrophoretic mobility shift assay (EMSA) as previously described (21–24) in a 15-µl binding reaction mixture containing 15 µg of nuclear proteins and 35 fmol of double-stranded NF-{kappa}B consensus oligonucleotide end labeled with [{gamma}-32P]ATP (Amersham; Piscataway, NJ) using T4 polynucleotide kinase (Promega). After incubation at room temperature for 20 min, the reaction mixture was separated on 5% nondenaturing polyacrylamide gels, and the density of the binding bands was established by densitometric analysis (Genomic Solutions; Ann Arbor, MI). The results from each group were expressed as relative integrated intensity compared with the normal heart group. A supershift assay using antibodies to P65 and P50 was performed to confirm NF-{kappa}B binding specificity as previously described (21–24).

Kinase activity assay. Kinase activity was measured as previously described (22, 24). Briefly, ~200 µg of cellular proteins were immunoprecipitated with 2 µg of antibodies against IKK-{beta} (Santa Cruz Biotechnology; Santa Cruz, CA) at 4°C for 1 h followed by the addition of 10 µl of protein A-agarose beads for another 1 h at 4°C. After being washed three times, the immunoprecipiates were resuspended in 15 µl of kinase buffer and subjected to kinase activity assay with 1 µg of glutathione S-transferase I{kappa}B-{alpha} substrate and 5 µCi of [{gamma}-32P]ATP (6,000 Ci/mmol, Amersham) at 30°C for 30 min. After the reactions were stopped by the addition of 3x Laemmli loading buffer, the reaction mixtures were resolved on polyacrylamide gels followed by autoradiography to Kodak X-ray films. The phosphorylation of substrate was quantified by scanning densitometry.

Western blot analysis. Cytoplasmic proteins (60 µg) were separated by SDS-PAGE and transferred onto Hybond ECL membranes (Amersham) as previously described (21–24). The membranes were incubated with rabbit anti-I{kappa}B-{alpha} or anti-atrial natriuretic peptide (ANP), full length (Santa Cruz Biotechnology), respectively, followed by secondary goat anti-rabbit IgG-conjugated peroxidase (Sigma Chemical; St. Louis, MO). The membranes were analyzed by the ECL system (Amersham Pharmacia). The signals were quantified by scanning densitometry and computer-assisted image analysis.

RT-PCR assay. Total RNA was isolated from rat myocardium using the Ultraspec-II RNA Isolation System (Biotecx Laboratories; Houston, TX), and RT-PCR assays were performed with RNA PCR kits (Perkin-Elmer Cetus; Norwalk, CT) as previously described (21–24). The ANP upstream primer was 5'-GTGACGGCTGAGGTTGTTTT-3', and the downstream primer was 5'-TTTGTGCTGGAAGATAAGAAA-3'. The brain natriuretic peptide (BNP) upstream primer was 5'-GACGGGCTGAGGTTGTTTTA-3' and the downstream primer was 5'-TTGTGCTGGAAGATAAGAAA-3'. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) upstream primer was 5'-TGAAGGTCGGTGTGAACGGATTTGG-3', and the downstream primer was 5'-ACGACATACTCAGCACCGGCCTCAC-3'. The PCR products of ANP, BNP, and GAPDH were analyzed by electrophoresis on 1% agarose gels (FMC Products), which were stained with ethidium bromide and scanned (Genomic Solutions). All values obtained with the ANP or BNP primers were normalized to the values obtained with the GAPDH primers. The results are expressed as the relative integrated intensity.

Histological examination. Three weeks after being aortic banded with or without treatment, the hearts were harvested, sectioned, and immersion fixed in 4% buffered paraformaldehyde from sham, aortic-banded, and treatment groups with 4 hearts/group. The tissues were embedded in paraffin, cut at 5 µm, counterstained with hematoxylin and eosin (H&E), and examined with brightfield microscopy.

Statistical analysis. Results are expressed as means ± SE. For tests of significance between the groups, one-way ANOVA was performed. P < 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Aortic banding induces cardiac hypertrophy. Figure 1 shows the HW/BW in rats at 1, 3, and 5 days and 1–6 wk after aortic banding and age-matched sham controls. HW/BW in aortic- banded rats was not markedly changed at 1 and 3 days but significantly increased by 19.1% at 5 days and by 35.8% at 1 wk, peaked at 4 wk (53.6%), and remained elevated at 6 wk after aortic banding compared with age-matched sham controls. The increased HW/BW was due to an increase in left ventricular weight, and there were no differences in body weight compared with age-matched sham controls.



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  		Fig. 1. Aortic banding for 1-, 3-, and 5-day (d) and 1–6 wk induced cardiac hypertrophy. Age-matched sham-operated rats served as controls. Left: representative heart showing that aortic banding induced cardiac hypertrophy. The arrow indicates the position of the aortic banding suture. Right: ratio of heart weight to body weight (HW/BW). N, normal hearts; sham, sham-operated controls; banding, aortic-banded hearts. There were 8–10 rats/group. *P < 0.05 compared with age-matched sham-operated controls.

 
Aortic banding increases ANP and BNP mRNA expression. The expressions of ANP and BNP mRNA are the established markers for the development of cardiac hypertrophy. Therefore, we examined the levels of ANP and BNP mRNA by RT-PCR in the rat hearts subjected to aortic banding for 1–6 wk, and age-matched sham-operated rats served as controls with 5–6 rats/group. As shown in Fig. 2A, the levels of ANP mRNA were significantly increased by 122.0% at the first week and peaked at the third week (277.2%), and the increased ANP mRNA persisted to the sixth week of aortic banding compared with age-matched sham controls. Figure 2B shows that the levels of BNP mRNA were also significantly increased at the first week, peaked at the second week, and persisted to the sixth week after aortic banding compared age-matched sham controls. A representative RT-PCR result is shown in Fig. 2C.



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  		Fig. 2. Aortic banding for 1–6 weeks increased atrial natriuretic peptide (ANP; A) and brain natriuretic peptide (BNP; B) mRNA expressions in rat hearts. The levels of ANP, BNP, and GAPDH mRNA in the hearts were examined by RT-PCR, and the expressions of ANP and BNP were expressed as relative ratios to GAPDH mRNA levels. *P < 0.05 compared with age-matched, sham-operated controls. C: representative RT-PCR result. M, 100-bp ladder. There were 5–6 rats/group.

 
Increased NF-{kappa}B binding activity in hypertrophic hearts. To investigate the time course of NF-{kappa}B activation during the development of cardiac hypertrophy, rats were subjected to aortic banding or sham surgical operation for 1, 3, and 5 days and 1–6 wk with 8–10 rats/group. The nuclear proteins were isolated from the hearts and subjected to EMSA for NF-{kappa}B activity. As shown in Fig. 3, NF-{kappa}B binding activity is low in normal hearts but significantly increased by 83.2% at the first day and peaked at the third week after aortic banding. The increased NF-{kappa}B binding activity in the aortic-banded hearts was maintained at the sixth week compared with age-matched sham controls. There was no significant change in NF-{kappa}B binding activity in the groups of age-matched sham controls. The specificity of NF-{kappa}B binding activity in the hypertrophic hearts was confirmed by the addition of 100-fold excess of unlabeled NF-{kappa}B or activator protein-1 oligonucleotides into the EMSA reaction (Fig. 3C). Unlabeled NF-{kappa}B oligonucleotides competed for the binding proteins in nuclear extracts prepared from aortic constricted rat hearts, whereas the unrelated activator protein-1 oligonucleotides did not. The predominant protein complex of NF-{kappa}B containing P50 and P65 subunits in the hypertrophic hearts was demonstrated by antibody supershift assays. Both antibodies considerably shifted the major NF-{kappa}B binding complex.



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  		Fig. 3. Increased NF-{kappa}B activation in hearts subjected to aortic banding for 1, 3, and 5 days and 1–6 wk. A: NF-{kappa}B activity was determined by electrophoretic mobility shift assay (EMSA) with nuclear extracts isolated from aortic-banded hearts and age-matched sham control hearts with 8–10 rats/group. *P < 0.05 compared with respective controls. B: representative EMSA of NF-{kappa}B activity. NF-{kappa}B binding bands are labeled on the right. nb, Nonspecific banding. C: specificity of NF-{kappa}B binding activity was analyzed by the addition of unlabeled oligonucleotides and by an antibody supershift gel assay. NF-{kappa}B banding activity was examined from heart samples subjected to aortic banding for 3 wk. NF-{kappa}B and the nonspecific binding band are labeled on the left, and the raised bands supershifted by antibodies (Ab) are indicated on the right. Free probe is not shown.

 
Increased IKK-{beta} activity in hypertrophic hearts. Because IKKs are important kinases for I{kappa}B-{alpha} phosphorylation and degradation, which is a key step for NF-{kappa}B activation, we examined IKK-{beta} activity in hearts from rats subjected to aortic banding or sham surgical operation for 1, 3, and 5 days and 1–6 wk with 5–6 rats/group. As shown in Fig. 4, the levels of IKK-{beta} activity were low in the normal hearts but significantly increased at the first day (421.3%), reached a peak at the fifth day (1,418%), and remained increased to the sixth week after aortic banding compared with age-matched sham controls.



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  		Fig. 4. Increased I{kappa}B kinase (IKK)-{beta} activity in hearts subjected to aortic banding for 1, 3, and 5 days and 1–6 wk. Cytoplasmic proteins were isolated from the hearts, and IKK-{beta} activity was analyzed by kinase assay with glutathione-S-transferase-I{kappa}B-{alpha} as the substrate. There were 5–6 rats/group. *P < 0.05 compared with age-matched sham controls.

 
Inhibition of NF-{kappa}B activation attenuates cardiac hypertrophy. I{kappa}B-{alpha}M is a superrepressor of NF-{kappa}B activation. To determine whether inhibition of NF-{kappa}B activation could attenuate the development of cardiac hypertrophy in vivo, we transfected the myocardium with Ad5-I{kappa}B-{alpha}M or Ad5-GFP immediately followed by banding the aorta for 3 wk. First, we examined the transfection efficiency by transfecting Ad5-I{kappa}B-{alpha}M or Ad5-GPF into the hearts. After 3 and 11 days of transfection of Ad5-GFP, the transfected hearts were harvested, sectioned, and examined under fluorescent microscopy. As shown in Fig. 5A, GFP expression was mainly located in cardiac myocytes and endothelial cells 3 days after transfection (a and b). GFP expression was also observed in cardiac myocytes and endothelial cells 11 days after transfection (Fig. 5A,c and d) but less than that after 3 days of transfection. Figure 5B shows higher levels of I{kappa}B-{alpha} present in heart samples transfected with Ad5-I{kappa}B-{alpha}M for 3 days. The levels of I{kappa}B-{alpha} in the myocardium transfected with Ad5-I{kappa}B-{alpha}M were still higher than those of nontransfected hearts 3 wk after transfection. Next, we examined the effects of Ad5-I{kappa}B-{alpha}M on the development of cardiac hypertrophy in vivo immediately followed by banding the aorta. Figure 6A shows that transfection of Ad5-I{kappa}B-{alpha}M significantly reduced the aortic banding-increased HW/BW by 64%, whereas control Ad5-GFP did not reduce HW/BW. Figure 6B shows that transfection of Ad5-I{kappa}B-{alpha}M also significantly blunted, but did not completely inhibit, aortic banding-increased NF-{kappa}B activity by 81% in hearts subjected to 3 wk of aortic banding. Transfection of Ad5-I{kappa}B-{alpha}M into the myocardium also significantly reduced aortic banding-increased levels of myocardial ANP by 47% compared with aortic banding group (Fig. 6C).



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  		Fig. 5. In vivo transfection of Ad5-green fluorescent protein (GFP) or Ad5-I{kappa}B-{alpha}M (I{kappa}B-{alpha}M) into rat hearts. Rat hearts were directly transfected with Ad5-GFP or an Ad5-I{kappa}B-{alpha} mutant [1 x 1010 plaque-forming units (pfu)/ml]. A: 3 (a and b) and 11 days (c and d) after the transfection of Ad5-GFP, hearts were harvested, sectioned, and visualized with white light (a and c) and fluorescent light (b and d) under microscopy. As shown in b and d, both myocytes and endothelium were labeled with Ad5-GFP. Magnification: x10. B and C: expression of transfected Ad5-I{kappa}B-{alpha} in the hearts. Three days (B) and 3 wk (C) after the transfection, I{kappa}B-{alpha} levels in the hearts were examined by Western blot analysis.

 


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  		Fig. 6. Transfection of I{kappa}B-{alpha}M attenuates cardiac hypertrophy, decreases NF-{kappa}B activity, and improves morphology of hypertrophic hearts in vivo. Rat hearts were directly transfected with Ad5-GFP or an Ad5-I{kappa}B-{alpha} mutant (1 x 1010 pfu/ml) immediately followed by banding the aorta. A: 3 wk after the aortic banding, hearts were harvested and HW/BW was analyzed. Inset, representative photograph of hearts. There were 8–10 rats/group. B: NF-{kappa}B activity was assessed by EMSA with nuclear extracts isolated from the hearts. Inset, representative EMSA result. NF-{kappa}B banding bands are labeled on the right. There were 8–10 rats/group. *P < 0.05 compared with age-matched sham-operated controls (S); #P < 0.05 compared with the aortic-banded group (B). C: administration of Ad5-I{kappa}B-{alpha}M or pyrrolidinedithiocarbamate (PDTC) reduced aortic banding-increased myocardial ANP. After 3 wk of aortic banding with and without administration of Ad5-I{kappa}B-{alpha}M or PDTC, the hearts were harvested and cellular proteins were prepared for Western blot analysis of ANP. *P < 0.05 compared with age-matched sham controls; #P < 0.05 compared with the aortic-banded group. There were 6–8 rats/group. D: transfection of Ad5-I{kappa}B-{alpha}M improved morphology of hypertrophic hearts. After 3 wk of aortic banding with and without transfection of Ad5-I{kappa}B-{alpha}M, the hearts were harvested, sectioned, embedded in paraffin, and counterstained with hematoxylin and eosin with 4 hearts/group. Representative phtomicrographs demonstrating morphology of heart sections from sham-operated (a–c), aortic-banded (d–f), and aortic-banded rats transfected with Ad5-I{kappa}B-{alpha}M (g-i) are shown. Magnification: x4, x10, and x20, respectively.

 
Transfection of I{kappa}B-{alpha}M improves morphology of hypertrophic hearts. To evaluate whether transfection of adenovirus causes cytotoxicity and induces an inflammatory response in the organ, we examined heart tissue sections transfected with Ad5-GFP for 3 and 11 days. We observed a mild inflammatory response around arteries within the myocardium (Fig. 5C, H&E staining). Using high magnification, we observed that most of the infiltrating cells were mononuclear cells. To determine whether transfection of Ad5-I{kappa}B-{alpha}M affects cardiac myocyte death or survival in hypertrophic hearts, we transfected Ad5-I{kappa}B-{alpha}M into the myocardium immediately followed by aortic banding for 3 wk. We observed that the hypertrophic hearts induced by aortic banding exhibited extensive cardiac myocyte death and loss and increased myocardial fibrosis (Fig. 6D). Transfection of Ad5-I{kappa}B-{alpha}M into the myocardium, however, reduced cardiac myocyte death and loss compared with aortic banding without treatment (Fig. 6D).

Antioxidant PDTC administration attenuates cardiac hypertrophy. Recent studies suggested that reactive oxygen species (ROS) are involved in the hypertrophic response of cardiomyocytes (1, 15). Because ROS can significantly induce NF-{kappa}B activation (3), we examined the effect of systemic administration of the antioxidant PDTC, which can inhibit NF-{kappa}B activation, on the development of cardiac hypertrophy in vivo. We administered PDTC into the rats by daily intraperitoneal injection (80 mg·kg–1·day–1) for 3 wk after aortic banding. As shown in Fig. 7A, administration of PDTC significantly reduced the aortic banding-increased HW/BW (48%) in vivo, indicating a reduction in the development of cardiac hypertrophy. Figure 7B shows that PDTC treatment significantly blunted NF-{kappa}B activity (52%) compared with untreated aortic-banded rats. As shown in Fig. 6C, PDTC administration also significantly reduced aortic banding-increased myocardial ANP levels by 51% compared with the aortic-banded group.



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  		Fig. 7. PDTC administration attenuates cardiac hypertrophy and decreases NF-{kappa}B activity in vivo. PDTC was administered by an intraperitoneal injection to rats for 3 wk after aortic banding. A: HW/BW. Inset, representative photograph of hearts. B: NF-{kappa}B activity was assessed by EMSA with nuclear extracts isolated from the hearts. Inset, representative EMSA result. NF-{kappa}B banding bands are labeled on the right. There were 8–10 rats/group. *P < 0.05 compared with age-matched sham-operated controls; #P < 0.05 compared with the aortic banding group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
A significant finding in the present study is that NF-{kappa}B activity was increased in rat hearts subjected to aortic banding. Inhibiting NF-{kappa}B activation by in situ transfection of an I{kappa}B-{alpha} dominant negative mutant, a superrepressor of NF-{kappa}B activation, or by systemic administration of PDTC, an antioxidant that has been shown to inhibit NF-{kappa}B activation, significantly attenuated the development of cardiac hypertrophy in vivo. Our results suggest that NF-{kappa}B activation is required for the development of cardiac hypertrophy in vivo.

We have previously shown the activation of myocardial NF-{kappa}B by I/R injury both in vitro and in vivo in rat hearts (21–24). Because cardiac hypertrophy is an adaptive response to various stimuli, including myocardial I/R injury and pressure overload, it is possible that NF-{kappa}B activation may participate in the development of cardiac hypertrophy in vivo. In the present study, we observed that NF-{kappa}B activation was increased in aortic banding-induced hypertrophic hearts. Our in vivo observation is consistent with observations from in vitro studies. For example, ANG II stimulated the hypertrophic response of cardiomyocytes, and ANP gene expression requires NF-{kappa}B activation (25, 33). Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) activators inhibited the hypertrophic response of cardiomyocytes in vitro by preventing NF-{kappa}B activation (37). Antioxidants can abolish the ANG II-induced hypertrophic response of cardiomyocytes through inhibiting NF-{kappa}B activation (17). Increased NF-{kappa}B activation was also observed in the myocardium of patients with congestive heart failure (10, 36).

Aortic banding is a well-established model system to evaluate the development of left ventricular hypertrophy in response to hemodynamic overload. The molecular mechanisms by which pressure overload induces cardiomyocyte hypertrophy, however, are poorly understood. It is generally believed that a mechanical signal induced by pressure overload will initiate a cascade of biological signal transduction pathways, leading to an increase in protein synthesis and cardiac growth (26). It has been reported that ANG II plays a mandatory role in the induction of the hypertrophic response via an AT1 receptor-mediated signaling pathway, but recent studies using AT1a receptor knockout mice suggest that ANG II is not mandatory for load-induced hypertrophy (13). Although we did not observe a significant change in the levels of myocardial ANG II in hypertrophic hearts (data not shown), myocardial NF-{kappa}B activation was significantly increased in aortic banded hearts. Blunting NF-{kappa}B activation by transfection of Ad5-I{kappa}B-{alpha}M attenuated aortic banding-induced cardiac hypertrophy in vivo and prevented cardiac myocyte death and loss in the hypertrophic hearts, suggesting that NF-{kappa}B activation participates in the development of cardiac hypertrophy.

I{kappa}B-{alpha}M is an I{kappa}B-{alpha} dominant negative mutant with a serine-to-alanine mutation at amino acids 32 and 36 and is a superrepressor of NF-{kappa}B activation that prevents signal-induced phosphorylation and subsequent proteosome-mediated degradation of I{kappa}B-{alpha}. E1 and E3 genes (Ad5) expressing I{kappa}B-{alpha}M (Ad5-I{kappa}B-{alpha}M) were employed to inhibit NF-{kappa}B activation because the recombinant adenoviruses expressing genes of interest result in a high-transfection efficiency in vivo (20) and are currently the most often employed for gene transfer in cardiovascular tissues. It has been reported, however, that transfection of recombinant adenoviruses can cause significant cytotoxicity and immunological responses (5). In the present study, we observed a mild inflammatory response in the myocardium transfected with Ad5-GFP, and most of these infiltrating cells were mononuclear cells around arterioles. Our observation is similar to previous studies that directly transfected adenovirus into rat hearts (7), indicating that the cytotoxicity caused by transfected adenoviruses may be related to different organs transfected and different delivery approaches employed. Interestingly, there was abundant death and loss of cardiac myocytes in the hypertrophic hearts induced by aortic banding for 3 wk. Transfection of Ad5-I{kappa}B-{alpha}M reduced cardiac myocyte death and loss, suggesting that prolonged NF-{kappa}B activation in the hypertrophic hearts may be involved in stimulating the expression of pro-cell death genes.

Oxidative stress or ROS are involved in the hypertrophic response of cardiomyocytes (1, 15). Because ROS are a strong stimulus for activation of NF-{kappa}B (3), we examined whether antioxidants could ameliorate cardiac hypertrophy through inhibiting NF-{kappa}B activation. We observed that administration of the antioxidant PDTC into aortic-banded rats significantly attenuated the development of cardiac hypertrophy in vivo with concomitant blunting of NF-{kappa}B activation. PDTC has been extensively documented to inhibit NF-{kappa}B activation, and its inhibitory effect has been attributed to its antioxidant properties (19) and its inhibition of the I{kappa}B-ubiquitin ligase activity toward phosphorylated I{kappa}B-{alpha} (14). In vivo administration of PDTC at doses of 50–200 mg·kg–1·day–1 significantly inhibited NF-{kappa}B activation and inflammatory cytokine production in the myocardium (30). In the present study, we observed that systemic administration of PDTC by intraperitoneal injection at a dose of 80 mg·kg–1·day–1 for 3 wk reduced the development of cardiac hypertrophy in vivo. In vitro studies have shown that prevention of NF-{kappa}B activation, by antioxidants or an I{kappa}B-{alpha} dominant negative mutant, ameliorated ANG II- or cardiotrophin-1-induced hypertrophic responses of cardiac myocytes (33). A recent study showed that aortic banding rapidly and transiently induced the expression of A20, a specific feedback inhibitor of NF-{kappa}B activation (4). Transfection of adenovirus expressing A20 (Ad5-A20) into cultured cardiac myocytes significantly inhibited the hypertrophic response of the cardiomyocytes to the stimulation with phenylephrine or endothelin-1 without resulting in cardiomyocyte apoptosis (6). Collectively, our results suggest that NF-{kappa}B could be a target for preventing cardiac hypertrophy in vivo.

A role of NF-{kappa}B in antiapoptosis and pro-cell death has been well documented. Complete inhibition of NF-{kappa}B activation could result in cardiac apoptosis in vitro (4). In the present study, we did not observe significant toxicity, as indicated by lethality, loss of body weight, and cardiac myocyte apoptosis examined by the TUNEL assay on heart tissue sections (data not shown) after the administration of either Ad5-I{kappa}B-{alpha}M and PDTC. The doses of Ad5-I{kappa}B-{alpha}M and PDTC chosen for the present study were based on our preliminary observation that Ad5-I{kappa}B-{alpha}M or PDTC effectively blunts, but does not completely inhibit, myocardial NF-{kappa}B activation. Maintaining certain levels of myocardial NF-{kappa}B activation, rather than completely inhibit its activity, may be beneficial to the treatment for reducing hypertrophic response of cardiac myocytes without inducing cardiac myocyte apoptosis.

In the present study, we observed that NF-{kappa}B activation was significantly increased in hypertrophic hearts and blunting NF-{kappa}B activation either by transfecting Ad5-I{kappa}B-{alpha}M or administration of the antioxidant PDTC significantly reduced the development of cardiac hypertrophy in vivo. NF-{kappa}B activation can be stimulated by a number of signaling pathways, including protein kinase C, PI3K/Akt, calcium/NFAT, Ca2+-calmodulin kinase IV, Ras, gp130, and JAK/STAT-mediated signaling pathways, which have been documented to play a role in cardiac hypertrophic responses (16, 29, 31, 34, 35). However, recent studies have highlighted signaling mediated by Toll-like receptors (TLRs), which activate NF-{kappa}B through MyD88-dependent pathways (27). Activation of TLR/MyD88-dependent signaling intermediates, such as TLR4 (9), TAK1 (39), and IKK-{beta} (33), has been shown to be involved in cardiac hypertrophy and heart failure. Elucidation of a role of TLR/MyD88-dependent NF-{kappa}B activation signaling could provide significant insights into the mechanisms of cardiac hypertrophy in vivo.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This study was supported by American Heart Association Grants 0051480B and 0255038B, National Institutes of Health (NIH) Grant RO1 HL-071837, and East Tennessee State University Research Development Committee (to C. Li); NIH Grants GM-53552, A-145829, and AT-00501 (to D. L. Williams); and National Gongguan Project of China Grant 2001BA710B (to X. Gao).


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Li, Dept. of Surgery, James H. Quillen College of Medicine, East Tennessee State Univ., Campus Box 70575, Johnson City, TN 37614-0575 (E-mail: Li{at}mail.etsu.edu)

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
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 REFERENCES
 

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