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Am J Physiol Heart Circ Physiol 289: H20-H29, 2005. First published March 4, 2005; doi:10.1152/ajpheart.00082.2005
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Inhibition of NF-{kappa}B induces regression of cardiac hypertrophy, independent of blood pressure control, in spontaneously hypertensive rats

Sudhiranjan Gupta, David Young, and Subha Sen

Department of Molecular Cardiology, Cleveland Clinic Foundation, Cleveland, Ohio

Submitted 27 January 2005 ; accepted in final form 1 March 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The transcription factor nuclear factor (NF)-{kappa}B plays a leading role in cardiac hypertrophy associated with heart failure, but whether it is involved in cardiac mass reduction is not known. We evaluated whether inhibiting the NF-{kappa}B cascade with pyrrolidine dithiocarbamate (PDTC) in spontaneously hypertensive rats (SHRs) and age-matched Wistar-Kyoto rats (WKYs) affected hypertrophy. We measured NF-{kappa}B signaling components [NF-{kappa}B translocation, I{kappa}B{alpha}, p65, mRNA and protein levels, and I{kappa}B kinase-{beta} (IKK{beta}) activity] at 12 and 36 wk in WKYs and SHRs and at 10 wk in PDTC-treated rats (n = 9). NF-{kappa}B activation was also evaluated in rats treated for 10 wk with captopril or hydralazine alone or with either drug plus PDTC. All components were increased in SHRs compared with WKYs. After PDTC treatment, NF-{kappa}B activity was inhibited, and heart weight-to-body weight ratio in SHRs was significantly attenuated (3.52 ± 0.04 to 3.32 ± 0.05 mg/kg). Captopril treatment significantly reduced cardiac mass (3.5 vs. 3.05 mg/kg; n = 9) and inhibited NF-{kappa}B activity (169.71 ± 5.70 to 106.7 ± 12.44). Hydralazine had no effect on cardiac mass (3.5 vs. 3.42 mg/kg) or NF-{kappa}B activity (169.71 ± 5.70 to 155.52 ± 6.11). Hydralazine plus PDTC reduced blood pressure (191.16 ± 1.7 to 158.5 ± 2.36 mmHg) and inhibited NF-{kappa}B activity (169.71 ± 5.70 to 97.29 ± 3.65). Our data suggest that 1) cardiac hypertrophy in SHRs is partly due to NF-{kappa}B activation, 2) inhibition of NF-{kappa}B activity by PDTC parallels regression of hypertrophy, and 3) regression of hypertrophy is partly due to inhibition of NF-{kappa}B activity, independent of hypertension. The relationship between NF-{kappa}B activity and cardiac remodeling is causal, not coincidental.

nuclear factor-{kappa}B; pyrrolidine dithiocarbamate; captopril; hydralazine

NUCLEAR FACTOR (NF)-{kappa}B is a well-characterized transcription factor that elicits a hypertrophic response in cardiac myocytes (10). It is a multifunctional ubiquitous transcription factor that has been shown to modulate a variety of gene expressions encoding the transcription of inflammatory genes, such as cytokines, chemokines, and growth factors in the diverse biological arena (1). In resting cells, NF-{kappa}B proteins are present in the cytoplasm as inactive heterodimers composed of two subunits p50 and p65 and are bound to the inhibitory protein I{kappa}B{alpha}, which prevents it from translocating into nucleus of the cell (2). On stimulation, I{kappa}B is phosphorylated and proteolytically degraded through a 26S proteasome-mediated pathway, which facilitates NF-{kappa}B to translocate into the nucleus and regulate gene transcription (2, 39). A critical step in the activation of NF-{kappa}B is the phosphorylation of I{kappa}Bs by a multimeric complex called I-{kappa}B kinase (IKK) complex (5), which consists of two catalytic subunits (IKK{alpha} and IKK{beta}) (45), the NF-{kappa}B essential modulator (44), IKK{gamma} (29), and IKK-associated protein (20). A general paradigm for the activation of NF-{kappa}B by the IKK complex supports that signal-induced activation of the I{kappa}B kinases phosphorylates I{kappa}B{alpha}, which triggers subsequent I{kappa}B{alpha} degradation and NF-{kappa}B activation/translocation. Biochemical and genetic ablation studies indicate that IKK{beta} may be more important in controlling NF-{kappa}B activation than IKK{alpha} (17–19). Previously, we and others have shown the activation of NF-{kappa}B during the cardiac hypertrophic process in neonatal cardiac myocytes (10, 25). Recently, Li et al. (16) have shown that treatment with PDTC, as a preventive measure, resulted in attenuation of hypertrophy in a aortic banding model. To evaluate whether this NF-{kappa}B activation also occurs during the chronic phase of hypertrophy in other hypertrophic models, we used the spontaneously hypertensive rat (SHR), a genetic model for cardiac hypertrophy. The SHR is a model of chronic pressure overload associated with compromised cardiac function (41). We also studied the effect of inhibition of NF-{kappa}B cascade by PDTC and determined its effect on cardiac mass in the presence and absence of hypertrophy.

In the present study, we sought to investigate the activation of NF-{kappa}B during the progression of cardiac hypertrophy in SHR and to further evaluate a cause and effect relationship between cardiac mass and NF-{kappa}B activation cascade after inhibition of NF-{kappa}B activity by PDTC'. To evaluate the influence of hypertension, we examined the effect of captoril [a known angiotensin-converting enzyme (ACE) inhibitor that regresses cardiac hypertrophy and blood pressure] and hydralazine (a known vasodilator that reduces blood pressure but does not have any effect on cardiac mass) on NF-{kappa}B activation to establish a relationship between cardiac mass and NF-{kappa}B activation in SHR.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Model

Male SHR and normotensive Wistar-Kyoto rats (WKY) were purchased from Taconic (Germantown, NY) and boarded at the animal facility at the Cleveland Clinic Foundation until the time of study. All rats were housed under identical conditions and had free access to food and water ad libitium. All procedures were performed within the regulation of the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals with approval from the Institutional Animal Care and Use Committee of the Cleveland Clinic Foundation.

Treatment Protocol

Forty-five SHR at 12 wk of age and forty-five age-matched WKY were treated as the following groups: group I, SHR untreated (water); group II, SHR + PDTC (150 mg·kg–1·day–1); group III, SHR + captopril (25 mg·kg–1·day–1); group IV, SHR + hydralazine (25 mg·kg–1·day–1); group V, SHR + PDTC + hydralazine; group VI, WKY untreated (water); group VII, WKY + PDTC (150 mg·kg–1·day–1); group VIII, WKY + captopril (25 mg·kg–1·day–1); group IX, WKY + hydralazine (25 mg·kg–1·day–1); and group X, WKY + PDTC + hydralazine.

All drugs were given separately in drinking water with 3% sucrose for a period of 10 wk. Untreated group I (WKY and SHR) received standard chow and water with 3% sucrose.

At the end of the treatment period, the rats were euthanized by decapitation. The hearts were removed, with the blood squeezed out, washed in cold PBS, and weighed in an analytical balance.

Determination of Mean Systolic Blood Pressure

In all rats, arterial pressure was measured by the tail-cuff method using a RT-BP-1000 rat tail blood pressure machine (Harvard Apparatus). The blood pressure was measured twice a week in each rat by the same person and at the same time of day (35).

Cytoplasmic and Nuclear Protein Extracts

SHR and WKY rat hearts, treated and untreated, were washed with cold PBS and minced with a sterile blade. Nuclear and cytoplasmic extracts were made according to the method described previously by Dignam et al. (9). All buffers were kept on ice unless stated otherwise. Phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), and a protease inhibitor cocktail were added just before use. The cytoplasmic and nuclear extracts were normalized for protein amounts determined by Bradford assay using bovine serum albumin as a standard (Bio-Rad Protein Assay Kit). Protein fractions were aliquoted and stored at –70°C.

Determination of NF-{kappa}B Activation

Electrophoretic mobility shift assay. An electrophoretic mobility shift assay (EMSA) was performed by using a double-stranded NF-{kappa}B binding site oligonucleotide as a probe as described previously (10).

Western blot analysis. SHR and WKY rat hearts, both treated and untreated, were washed in cold PBS, minced with a sterile blade, and lysed on ice in a buffer containing 10 mM Tris·HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 10 mM sodium pyrophosphate, 10% glycerol, 50 mM sodium fluoride, 100 µM Na3VO4, 10 nM okadaic acid, 0.5 mM DTT, 1 mM PMSF, 1% Triton-X-100, 1 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 µg/ml pepstatin. The lysates were centrifuged at 14,000 g for 20 min at 4°C. The total protein concentration was measured by the Bradford method. Samples containing 50 µg of protein were separated on 10% SDS-polyacrylamide gels and were electrophorectically transferred onto polyvinylidene difluoride membrane by using a wet transfer apparatus (Bio-Rad). Membranes were incubated in a blocking buffer containing 5% nonfat dry milk (Bio-Rad) in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBS-T). Membranes were probed with phospho-I{kappa}B{alpha}, I{kappa}B{alpha}, I{kappa}B{beta}, and NF-{kappa}B p65 antibodies (Cell Signaling Technology, Beverly, CA) overnight at 4°C (all at 1:1,000 dilution), washed three times in TBS-T, and then detected by using a horseradish peroxidase-conjugated secondary antibody and ECL (NEN). Actin and histone antibodies (Santa Cruz Biotechnology) were used as an internal protein loading control for cytoplasmic and nuclear protein, respectively.

RNA extraction and Northern blot analysis. Total RNA was isolated from the hearts of SHR and WKY rats according to the protocol of Chomczynsky et al. (6). RNA was resuspended in diethylpyrocarbonate water and quantitated by optical density at 260 nm. Transcript levels of I{kappa}B{alpha} and p65 were determined by Northern blot analysis. Northern blotting and hybridization were performed as described previously (10). For I{kappa}B{alpha}, a cDNA probe was used, kindly given by Dr. Allan Braiser (University of Texas), and for p65, a cDNA probe was kindly given by Dr. Sankar Ghosh (Yale University).

Kinase activity assay. Approximately 500 µg of cytoplasmic protein from each treated and untreated SHR and WKY rat hearts were immunoprecipitated with IKK{beta} antibody and protein A-agarose at 4°C for 1 h. The kinase assay was performed by using GST-I{kappa}B{alpha} as a substrate, according to the protocol by Li et al. (15).

Statistical Analysis

Results are expressed as means ± SE. Differences between groups were tested for statistical significance by paired Student's t-test. For various group analyses, we used Graph-Pad Prism. Differences were considered significant at P < 0.001.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
NF-{kappa}B Activation Cascade During the Progression of Cardiac Hypertrophy in SHR

Increased level of NF-{kappa}B binding activity in SHR. To determine the NF-{kappa}B binding activity in SHR and WKY rat hearts, EMSA was performed in nine SHR and nine WKY rat hearts. The data are shown in Fig. 1A. At 12 wk of age a 3.26-fold and at 36 wk of age a 5.17-fold (P < 0.001) increase in NF-{kappa}B binding activity were observed in SHR hearts (Fig. 1A) compared with corresponding WKY rat hearts. The specific binding of NF-{kappa}B in SHR hearts was confirmed by the addition of a 100-fold molar excess of unlabeled NF-{kappa}B competitor DNA into the EMSA reaction. Unlabeled NF-{kappa}B DNA competed for binding in the extracts prepared from SHR hearts (Fig. 1A). The presence of NF-{kappa}B in the protein complex was also demonstrated by antibody supershift assays. The p65 antibody considerably shifted the major NF-{kappa}B binding complexes (Fig. 2A). Further characterization of NF-{kappa}B activation was performed by Western blot by using a p65-specific antibody. Figure 1B shows the Western blot profile of nuclear fractions from WKY and SHR hearts as described in Fig. 1A. Histone monoclonal antibody was used as an internal protein loading control. Figure 1C presents the fold value of DNA binding activity in SHR hearts compared with WKY rat hearts.



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  		Fig. 1. Nuclear factor (NF)-{kappa}B activation during progression of hypertrophy in spontanteously hypertensive rats (SHR). A: nuclear protein was extracted from the hearts of 12- and 36-wk-old Wistar-Kyoto (WKY) and SHR rats. Binding reactions or gel mobility shift assay (GMSA) was performed with an NF-{kappa}B oligonucleotide labeled with [32P]dATP. Complex formation was eliminated with excess unlabeled NF-{kappa}B oligonucleotide. Complex formation was further confirmed by supershift analysis by using p65 antibody. B: quantification of NF-{kappa}B binding in SHR compared with WKY rat hearts. Results from GMSA illustrated in A were quantified by video image analyzer. An arbitrary density unit of the image analyzer was used for this purpose. Values are means ± SE. C: Western blot profile of the translocation of NF-{kappa}B p65 protein into the nucleus of both WKY and SHR rat hearts. Histone antibody (HA) was used as an internal nuclear protein loading control. These results are presented as the mean ± SE and represent seven different experiments with different rats. P < 0.001 compared with the WKY rats.

 


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  		Fig. 2. Upregulation of I{kappa}B{alpha} protein during progression of hypertrophy in SHR. Cytoplasmic protein extracts were made from both WKY and SHR rat hearts at 12 and 36 wk of age. A: tissue extracts (50 µg) were analyzed for intracellular level of I{kappa}B{alpha} protein content using a kit from Cell Signaling Technology. Actin protein was used as an internal loading control. B: quantification of I{kappa}B{alpha} protein in SHR compared with WKY rat hearts. Results from Western blot analysis illustrated in A were quantified by video image analyzer. An arbitrary density unit of the image analyzer was used for this purpose. Values represent means ± SE and represent seven different experiments with different mice. P < 0.001 compared with the WKY rats.

 
Enhancement of total I{kappa}B{alpha} activity in SHR. To investigate the functional role of I{kappa}B{alpha} in SHR during the progression of hypertrophy, we performed Western blot analysis to find out the phosphorylation status as well as the expression level of I{kappa}B{alpha} protein. Our data showed a 2.16-fold (average in 12-wk-old SHR) and 3.64-fold (average in 36-wk-old SHR) (P < 0.001) increase of I{kappa}B{alpha} protein level in SHR hearts compared with the WKY rat hearts (Fig. 2A). Actin antibody was used as an internal protein loading control. Figure 2B presents the fold value of I{kappa}B{alpha} activity in SHR hearts compared with WKY rat hearts. We used nine SHR and nine WKY rat hearts, and all showed the similar pattern of expression.

Increased level of IKK{beta} activity in SHR hearts. Because NF-{kappa}B activity is largely governed by IKK{beta}-dependent phosphorylation of I{kappa}B{alpha}, we determined the IKK{beta} activity in SHR. Our data showed an increase level of IKK{beta} activity (2.26-fold at 12 wk and 4.34-fold at 36 wk, P < 0.001) in SHR hearts compared with WKY rat hearts. The result is shown in Fig. 3A and is the representative of nine SHR and seven WKY rat hearts. Figure 3B represents the fold value of IKK{beta} activity in SHR hearts compared with WKY rat hearts.



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  		Fig. 3. Enhanced I{kappa}B kinase {beta} (IKK{beta}) activity during progression of hypertrophy in SHR. Cytoplasmic protein extracts were made from both WKY and SHR rat hearts at 12, 16, and 36 wk of age. A: tissue extracts (500 µg) were immunoprecipitated with IKK{beta} antibody, and kinase activity was determined with GST-I{kappa}B{alpha} as a substrate. B: quantification of IKK{beta} activity in SHR compared with WKY rat hearts. Results from kinase assay illustrated in A were quantified by video image analyzer. An arbitrary density unit of the image analyzer was used for this purpose. Values represent means ± SE and represent seven different experiments with different mice. P < 0.001 compared with WKY rats.

 
Enhanced expression of I{kappa}B{alpha} and p65 mRNA. As we observed an upregulation of I{kappa}B{alpha} protein level in SHR hearts, we further determined the level of I{kappa}B{alpha} mRNA in SHR (n = 9) and WKY (n = 9) rat hearts. The data presented in Fig. 4A showed a 2.24-fold increase at 12 wk and 4.41-fold increase at 36 wk (P < 0.01) of I{kappa}B{alpha} mRNA in SHR heart samples compared with WKY rat hearts. Moreover, we also observed an upregulation of p65 mRNA (1.82-fold at 12 wk and 3.81-fold at 36 wk, P < 0.001) in SHR hearts compared with WKY rat hearts (Fig. 4C). Figure 4, B and D, represents the fold value of I{kappa}B{alpha} and p65 mRNA level, respectively, in SHR hearts compared with WKY rat hearts.



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  		Fig. 4. Increased mRNA levels of both I{kappa}B{alpha} and p65 during progression of hypertrophy in SHR. A: total RNA was extracted from hearts of 12- and 36-wk-old WKY and SHR rats as described in MATERIALS AND METHODS. I{kappa}B{alpha} mRNA expression was determined by using a cDNA probe for I{kappa}B{alpha} labeled with [32P]dCTP. B: quantification of I{kappa}B{alpha} mRNA in SHR compared with WKY rat hearts. Results from Northern blot analysis illustrated in A were quantified by video image analyzer. An arbitrary density unit of the image analyzer was used for this purpose. C: mRNA expression was determined by using a cDNA probe for p65 labeled with [32P]dCTP. D: quantification of p65 mRNA in SHR compared with WKY rat hearts. Results from Northern blot analysis illustrated in B were quantified by video image analyzer. An arbitrary density unit of the image analyzer was used for this purpose. Values represent means ± SE and represent seven different experiments with different rats.

 
Effect of PDTC, Captopril, PDTC + Hydralazine, and Hydralazine on Blood Pressure and Cardiac Mass in SHR and WKY Rats

As shown in Fig. 5A, the mean arterial blood pressure at 16 wk of age of SHR was attenuated in PDTC-treated animals (191.16 ± 1.7 vs. 172.8 ± 1.54 mmHg). At the same age, hydralazine treatment reduced blood pressure to 154 ± 2.4 mmHg and hydralazine + PDTC treatment reduced the pressure to 158.5 ± 2.36 mmHg. In addition, we observed a significant decrease in blood pressure level in the captopril-treated animals (124.6 ± 0.23 mmHg).



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  		Fig. 5. Effect of pyrrolidine dithiocarbamate (PDTC), captopril (Capto), hydralazine (Hydral), and PDTC + hydralazine on blood pressure and cardiac mass in SHR and WKY rats. A: blood pressure profile in PDTC, captopril, hydralazine, and PDTC + hydralazine in WKY and SHR. B: heart weight-to-body weight ratio in PDTC, captopril, hydralazine, and PDTC + hydralazine in WKY and SHR.

 
With regard to the regression of hypertrophy in PDTC-, captopril-, hydralazine-, and PDTC + hydralazine-treated animals, we determined their heart weight-to-body weight ratio. As shown in Fig. 5B, PDTC-treated SHR show a declination of heart weight-to-body weight ratio compared with untreated SHR (3.52 ± 0.04 vs. 3.32 ± 0.05). PDTC along with hydralazine showed similar results (3.31 ± 0.04), but hydralazine alone had no effect on it (3.52 ± 0.04 vs. 3.42 ± 0.05). On the other side, captopril significantly reduced the heart weight-to-body weight ratio (3.52 ± 0.04 vs. 3.05 ± 0.05) as expected.

Effect of PDTC Treatment on NF-{kappa}B Signaling Cascade in SHR

On NF-{kappa}B activation and I{kappa}B{alpha} protein level in SHR. Two groups of rats (n = 9) were treated with PDTC (150 mg·kg–1·day–1) for a period of 10 wk and were killed afterward. The result is shown in Fig. 6, A and B. PDTC treatment significantly reduced NF-{kappa}B binding activation in SHR (169.7 ± 5.7 to 93.06 ± 8.84, P < 0.001) but not in WKY. Binding activity was further confirmed by competition assay and supershift analysis as described above. Figure 6B presents the fold value of DNA binding activity in SHR hearts compared with WKY rat hearts. NF-{kappa}B binding activity was unchanged in PDTC-treated WKY.



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  		Fig. 6. PDTC treatment reduced NF-{kappa}B activation and attenuated I{kappa}B{alpha} protein content in SHR. A: nuclear proteins were extracted and binding reactions (GMSA) were performed as described in Fig. 1. B: quantification of NF-{kappa}B binding of PDTC-treated SHR hearts. Results from GMSA illustrated in A were quantified by video image analyzer. An arbitrary density unit of the image analyzer was used for this purpose. Values represent means ± SE. *P < 0.001 compared with untreated SHR. C: cytoplasmic extracts made from WKY, SHR, and PDTC-treated SHR, and Western blot analysis was performed as described in Fig. 2. Results from Western blot analysis illustrated in B were quantified by video image analyzer. An arbitrary density unit of the image analyzer was used for this purpose. D: quantification of cytoplasmic protein level of PDTC-treated SHR. Values represent means ± SE and represent seven different experiments with different rats. *P < 0.001 compared with untreated SHR.

 
The appearance of total I{kappa}B{alpha} cytosolic protein was investigated by immunoblot analysis. The data are shown in Fig. 6, C and D. PDTC treatment significantly reduced the I{kappa}B{alpha} protein level in SHR (110.8 ± 3.36 to 59.4 ± 2.1, P < 0.001). Altogether, our data indicated that PDTC treatment significantly inhibited NF-{kappa}B activation and I{kappa}B{alpha} upregulation in SHR during the progression of hypertrophy. I{kappa}B{alpha} levels remain unchanged in PDTC-treated WKY (Table 1).


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  		Table 1. Effects of PDTC treatment on NF-{kappa}B components in WKY and SHR hearts

 
Effect of PDTC on IKK{beta} levels in SHR. To find out the effect of PDTC on IKK{beta} activation during the progression of hypertrophy, immunocomplex kinase assay was performed to detect the IKK{beta} activity in the SHR heart. The result is shown in Fig. 7A. PDTC treatment significantly inhibited IKK{beta} activity (40.0 ± 1.1 to 19.1 ± 1.1, P < 0.001) in SHR. Figure 7B presents the fold value of IKK{beta} activity in SHR hearts compared with WKY rat hearts. IKK{beta} activity remained unchanged in PDTC-treated WKY rats (Table 1).



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  		Fig. 7. PDTC treatment decreased IKK{beta} activity in SHR. A: cytoplasmic extracts were made from WKY, SHR, and SHR-treated with PDTC, and immunocomplex kinase assay was performed as described in Fig. 3. B: quantification of IKK{beta} activity of PDTC-treated SHR hearts. Results from kinase assay illustrated in A were quantified by video image analyzer. An arbitrary density unit of the image analyzer was used for this purpose. Values represent means ± SE and represent seven different experiments with different rats. *P < 0.001 compared with untreated SHR.

 
Effect of PDTC on I{kappa}B{alpha} and p65 mRNA level in SHR. We also determined the expression level of both I{kappa}B{alpha} and p65 mRNA levels in PDTC-treated SHR hearts. The data are shown in Fig. 8, A and D. Our results indicated that PDTC treatment inhibited both I{kappa}B{alpha} (294.2 ± 4.3 to 160.2 ± 2.1, P < 0.001) and p65 (100.8 ± 4.9 to 51.8 ± 1.6, P < 0.001) mRNA levels. Figure 8, B and D, presents the fold value of I{kappa}B{alpha} and p65 mRNA leveld in SHR hearts compared with WKY rat hearts. Both I{kappa}B{alpha} and p65 mRNA levels were unchanged in PDTC-treated SHR (Table 1). The effects of PDTC treatment in WKY and SHR on NF-{kappa}B components are summarized in Table 1.



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  		Fig. 8. PDTC treatment attenuated transcriptional level of both I{kappa}B{alpha} and p65. Total RNA was made from WKY, SHR, and PDTC-treated SHR, and Northern blot hybridization was performed using I{kappa}B{alpha} (A) and p65 (C) cDNA as a probe as described in Fig. 4. B: quantification of I{kappa}B{alpha} mRNA level of PDTC-treated SHR hearts. Results from Northern blot analysis illustrated in A were quantified by video image analyzer. D: quantification of p65 mRNA level of PDTC-treated SHR. An arbitrary density unit of the image analyzer was used for this purpose. Values represent means ± SE and represent eight (for I{kappa}B{alpha}) and seven (for p65) different experiments with different rats.

 
Effect of Captopril, Hydralazine, and PDTC + Hydralazine on NF-{kappa}B Activation in SHR

Effect of captopril, hydralazine, and PDTC + hydralazine are shown in Fig. 9A. Captopril treatment normalized the blood pressure (191.16 ± 1.7 to 124.6 ± 0.23) and reduced NF-{kappa}B binding activity to 153.4 ± 2.5 to 134.2 ± 2.1 (P < 0.001).



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  		Fig. 9. Captopril treatment partially inhibited NF-{kappa}B binding in SHR. Nuclear extracts were made from PDTC-, captopril-, and hydralazine-treated SHR. A: binding reactions using 32p NF-{kappa}B as a probes were performed as described in Fig. 2. B: quantification of NF-{kappa}B binding of PDTC-, captopril-, and hydralazine-treated SHR hearts. Results from GMSA illustrated in A were quantified by video image analyzer. An arbitrary density unit of the image analyzer was used for this purpose. Values represent means ± SE and represent nine different experiments with different rats.

 
Hydralazine treatment alone lowered the blood pressure but did not show any inhibition of NF-{kappa}B activity (153.4 ± 2.5 to 155.0 ± 3.5, P = not significant), whereas PDTC with hydralazine showed a similar degree of inhibition of NF-{kappa}B (153.4 ± 2.1 to 113.0 ± 3.4, P < 0.001) (Fig. 9, A and B) despite normalization of blood pressure, suggesting the regression of cardiac mass was mediated through NF-{kappa}B and was independent of blood pressure.

Effect of Captopril, Hydralazine, and PDTC + Hydralazine on I{kappa}B{alpha} Protein Levels in SHR

Three groups of rat (n = 9) were treated with captopril (25 mg·kg–1·day–1), hydralazine (25 mg·kg–1·day–1), and PDTC plus hydralazine for a period of 10 wk and were euthanized afterward. The result is shown in Fig. 10A. Captopril treatment reduced the I{kappa}B{alpha} protein level (114.86 ± 3.65 to 82.46 ± 3.41, P < 0.001).



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  		Fig. 10. Captopril treatment partially inhibited I{kappa}B{alpha} protein content in SHR. Cytoplasmic extracts were made from PDTC-, captopril-, and hydralazine-treated SHR. A: Western blotting using I{kappa}B{alpha} antibody as a probe was performed as described in Fig. 2. B: quantification of Western blotting of PDTC-, captopril-, and hydralazine-treated SHR hearts. An arbitrary density unit of the image analyzer was used for this purpose. Values represent means ± SE and represent nine different experiments with different rats. *P < 0.001 compared with the untreated SHR.

 
In addition, it is also observed that hydralazine treatment did not show any inhibition in total content of I{kappa}B{alpha} protein level (114.86 ± 3.65 to 112.64 ± 4.80, P < 0.001) (Fig. 10, A and B), suggesting that NF-{kappa}B-mediated cardiac mass attenuation was independent of blood pressure.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we determined 1) NF-{kappa}B activation during the progression of cardiac hypertrophy from 12 to 36 wk of age in SHR, and 2) the effect of inhibition of NF-{kappa}B by PDTC on cardiac mass remodeling in SHR. In addition, this is the first in vivo study to demonstrate that captopril, an angiotensin-converting enzyme inhibitor, attenuated the cardiac mass possibly through a NF-{kappa}B-mediated signaling pathway in SHR. Our data showed that during progression of hypertrophy and hypertension, NF-{kappa}B activation occurs progressively. Treatment with PDTC inhibited NF-{kappa}B activity and regressed cardiac hypertrophy. PDTC treatment also lowered blood pressure from 190 to 175 mmHg. Furthermore, to evaluate the role of blood pressure, captopril treatment (that normalized blood pressure and cardiac mass as expected) significantly inhibited NF-{kappa}B activity as well. Treatment with hydralazine alone did not alter either cardiac mass or NF-{kappa}B activity, despite the normalization of blood pressure. Interestingly, when hydralazine was combined with PDTC, the degree of inhibition of NF-{kappa}B was similar to PDTC alone, suggesting normalization of blood pressure did not reduce NF-{kappa}B activity any further. These observations suggest that regression of cardiac mass is due to inhibition of NF-{kappa}B activity and independent of blood pressure control.

A number of studies indicated the role of NF-{kappa}B in various cardiac disorder states (21, 28, 43, 42). However, there is no report of activation of NF-{kappa}B during the progression of cardiac hypertrophy in SHR. In this report, we examined and determined the components of the NF-{kappa}B signaling cascade (NF-{kappa}B activation, I{kappa}B{alpha} protein and mRNA level, p65 protein and mRNA level and IKK{beta} activity) in SHR during the progression of cardiac hypertrophy (at 12 and 36 wk of age). Our data revealed that all of the NF-{kappa}B signaling components were upregulated/increased during the progression of hypertrophy in SHR (Figs. 2–5) compared with age- and sex-matched WKY.

To establish whether there is any an association between NF-{kappa}B activation and cardiac remodeling, we treated the SHR with NF-{kappa}B inhibitor PDTC. Our data showed that PDTC attenuates 1) the cardiac mass or heart weight-to-body weight ratio (Fig. 5A), 2) blood pressure (Fig. 5B), and 3) activation of NF-{kappa}B signaling cascades (Figs. 6–8). All these findings suggest that PDTC attenuates the degree of hypertrophy in SHR. Although the exact mechanism of action of PDTC is not well understood but has shown to be an antioxidant and paradoxically, the prooxidant and metal-chelating properties of PDTC could also be involved in its ability to inhibit NF-{kappa}B activation (24). In our study, the upregulation of I{kappa}B{alpha} that occur during progression of hypertrophy in SHR was prevented by PDTC treatment, suggesting that PDTC may also inhibit NF-{kappa}B activation via stabilization of I{kappa}B{alpha}. Further biological effects of PDTC that have been considered include the interference with reactive oxygen metabolism (31), the chelation of divalent metal ions (37), or the influence of thiol levels (23). Therefore, the cardiac remodeling in SHR may be due to intense antioxidant effect, and PDTC possibly plays a protective role in this event. Therefore, the most effective NF-{kappa}B inhibitor appears to be the pyrrolidine derivative of dithiocarbamate (PDTC) as a result of its ability to traverse the cell membrane and its prolonged stability in solution at physiological pH (40). Furthermore, PDTC has been extensively documented to inhibit NF-{kappa}B activation, its inhibitory role has been shown to mediate through its antioxidant properties (12), and its inhibition is mediated through the production of reactive oxygen species (26). The dithiocarbamate represents a class of antioxidants reported to be a potent inhibitor of NF-{kappa}B in vitro (32–34).

Recently, few studies also revealed that aortic banding rapidly and transiently induced the expression of A20, a feedback inhibitor of NF-{kappa}B activation (3, 7). Our studies showed that PDTC significantly inhibited the NF-{kappa}B signaling components associated with a partial regression of the heart weight-to-body weight ratio in SHR. Furthermore, we also observed a partial attenuation of the blood pressure level in PDTC-treated SHR, suggesting that PDTC may play a role in reducing blood pressure. Therefore, the improvement of either cardiac hypertrophy or hypertension could also be due to an antioxidant effect. These findings led us to conclude that there may be an association of NF-{kappa}B activation and cardiac mass remodeling. Collectively, our data suggest that NF-{kappa}B could be a target for preventing cardiac hypertrophy in vivo.

Our data also demonstrated that captopril, an angiotensin-converting enzyme inhibitor, would partially inhibite NF-{kappa}B activation. It has been reported by numerous investigators (8, 22, 27, 36) that captopril regresses cardiac hypertrophy and normalizes blood pressure, but this is the first report to show that captopril also inhibits NF-{kappa}B activity as well. Recently, it has been reported that angiotensin II could activate NF-{kappa}B in cardiac disease (4, 30, 38). In our study, treatment of SHR with the angiotensin-converting enzyme inhibitor captopril inhibits angiotensin II formation thereby in turn inhibits the NF-{kappa}B activation. These results, therefore, indicate that captopril ameliorates cardiac hypertrophy in SHR and may indicate a possible involvement of NF-{kappa}B. To elucidate further whether blood pressure may influence NF-{kappa}B activation, we used hydralazine, a known vasodilator reported to normalize the blood pressure level but have no effects on cardiac mass (11, 13, 14, 36). Our study showed that hydralazine normalized the blood pressure in SHR, as expected, but had no effect in either cardiac mass or NF-{kappa}B activation. However, when hydralazine was given in combination with PDTC, NF-{kappa}B activation was inhibited. This observation suggests that regression of cardiac hypertrophy by PDTC and blood pressure control are two independent processes. All together, our data suggest that inhibition of NF-{kappa}B or anti-NF-{kappa}B therapy would be a possible therapeutic target for the treatment of cardiac hypertrophy.

In summary, the present study demonstrates that PDTC significantly regresses cardiac mass by inhibiting NF-{kappa}B activation in SHR, although the precise mode of action remains to be further explored. Furthermore, our data showed an association between NF-{kappa}B activation and cardiac remodeling. It is of note that the activation of NF-{kappa}B is a common end point of various signal transduction pathways, including PKC, protein tyrosine kinase, phospholipase C, mitogen-activated kinase, and other signaling factors. Thus inhibiting of NF-{kappa}B by PDTC or other inhibitor may represent a novel therapeutic strategy for the treatment of cardiac hypertrophy.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-RO1 47794 (to S. Sen).


   ACKNOWLEDGMENTS
 
The authors acknowledge Dr. A. Brasier for I{kappa}B{alpha} cDNA, Dr. S. Ghosh for p65 cDNA, the expert secretarial help of Jane Rein and Lori Sims, and the editing assistance of Christine Kassuba.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Sen, Dept. of Molecular Cardiology, NB 50, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195 (E-mail: sens{at}ccf.org)

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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