Involvement of the NF-κB/Matrix Metalloproteinase Pathway in Cardiac Fibrosis of Mice Lacking Guanylyl Cyclase/Natriuretic Peptide Receptor A*

Mice carrying a targeted disruption of the Npr1 gene (coding for guanylyl cyclase/natriuretic peptide receptor A (NPRA)) exhibit increased blood pressure, cardiac hypertrophy, and congestive heart failure, similar to untreated human hypertensive patients. The objective of this study was to determine whether permanent ablation of NPRA signaling in mice alters the expression of matrix metalloproteinase (MMP)-2 and MMP-9 and pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), leading to myocardial collagen remodeling. Here, we report that expression levels of the MMP-2 and MMP-9 genes were increased by 3-5-fold and that the expression of the TNF-α gene was enhanced by 8-fold in Npr1 homozygous null mutant (Npr1-/-) mouse hearts compared with wild-type (Npr1+/+) control mouse hearts. Myocardial fibrosis, total collagen, and the collagen type I/III ratio (p < 0.01) were dramatically increased in adult Npr1-/- mice compared with age-matched wild-type counterparts. Hypertrophic marker genes, including the β-myosin heavy chain and transforming growth factor-β1, were significantly up-regulated (3-5-fold) in both young and adult Npr1-/- mouse hearts. NF-κB binding activity in ventricular tissues was enhanced by 4-fold with increased translocation of the p65 subunit from the cytoplasmic to nuclear fraction in Npr1-/- mice. Our results show that reduced NPRA signaling activates MMP, transforming growth factor-β1, and TNF-α expression in Npr1-/- mouse hearts. The findings of this study demonstrate that disruption of NPRA/cGMP signaling promotes hypertrophic growth and extracellular matrix remodeling, leading to the development of cardiac hypertrophy, myocardial fibrosis, and congestive heart failure.

sponses, all of which contribute to the regulation of blood pressure and blood volume homeostasis (1,2). ANP and BNP bind to guanylyl cyclase/natriuretic peptide receptor A (NPRA), which is considered a major natriuretic peptide receptor that synthesizes the intracellular second messenger cGMP (3). Mice carrying a targeted disruption of the Npr1 gene (encoding NPRA) exhibit hypertension, marked cardiac hypertrophy, and congestive heart failure, with sudden death after 6 months of age (4 -6). On the other hand, Npr1 gene-duplicated mice have stimulated levels of guanylyl cyclase activity and increased accumulation of intracellular cGMP in a gene dose-dependent manner and exhibit protection against high salt diets (7). In vitro studies have shown that the ANP/NPRA system exerts growth inhibitory effects on hypertrophic agonist-induced proliferation of cardiac myocytes (8,9), fibroblasts (10), and mesangial and human vascular smooth muscle cells (11,12). Furthermore, transgenic mice overexpressing ANP have smaller hearts compared with wild-type mice, and ANP gene delivery attenuates cardiac hypertrophy in spontaneously hypertensive rats (13). Nonetheless, the molecular mechanism by which the ANP/NPRA system exerts protective effects and regulates cardiac remodeling in disease state is not well understood.
Abnormal cardiac remodeling is characterized by structural rearrangements that involve myocyte hypertrophy, hyperplasia of fibroblasts, and disproportionate increases in extracellular matrix (ECM) collagen deposition, which lead to myocardial fibrosis (14). ECM collagen is an important determinant of myocyte shape and alignment and plays regulatory roles in transduction of contractile force into overall cardiac ejection. Thus, remodeling of the myocardial collagen matrix is critical in the development of ventricular diastolic and systolic dysfunctions (14). Cardiac fibroblasts are the major cell type responsible for the synthesis of fibrillar collagen (types I and III), and synthesis and degradation of collagen in the myocardium are tightly controlled. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are the major regulators of collagen synthesis and degradation in the heart (15). Recent studies indicate that abnormal remodeling of myocardial collagen is caused by dysregulation of MMPs and their endogenous inhibitors, TIMPs (15,16). Increased activity of MMPs or decreased levels of TIMPs have been reported in hypertrophied and failing hearts, implicating that both MMPs and TIMPs play critical roles in the process of ventricular remodeling (17). A number of factors have been linked to stimulation of fibroblast proliferation and collagen deposition in the heart, including vasoactive hormones, cytokines, and growth factors (18,19); however, the mechanism that inhibits collagen production in the heart is not well understood.
The ANP/NPRA system has been implicated as an antihypertrophic and anti-fibrotic protective mechanism that moderates the cardiac remodeling process (4,5). ANP and BNP have been shown to inhibit fibroblast proliferation (8), collagen synthesis, and MMP release via the cGMP-dependent pathway and to have a broad functional opposition to transforming growth factor-␤1 (TGF-␤1)-induced ECM protein synthesis in vitro (20,21). However, in vivo studies have not been carried out to examine the role of NPRA signaling in regulation of MMPs, TIMPs, and pro-inflammatory mediators. In this study, we have utilized the Npr1 gene-disrupted mutant mouse model to determine the role of NPRA signaling in expression and activation of specific hypertrophic marker genes, MMPs, TIMPs, and ECM proteins. To our knowledge, this is the first report demonstrating that permanent ablation of NPRA signaling in mice modulates cardiac MMPs, TIMPs, and collagen remodeling, which play critical roles in cardiac hypertrophy and heart failure.
Generation of Mice and Genotyping-Npr1 gene-disrupted mice were generated by homologous recombination in embryonic stem cells as described previously (4,7). Animals were bred and maintained at the animal facility of the Tulane University Health Sciences Center. The mouse colonies were housed under 12-h light/dark cycles at 25°C and fed regular chow (Purina Laboratory) and tap water ad libitum. All animals were littermate progenies of the C57/BL6 genetic background and were designated as Npr1 gene-disrupted homozygous null mutant (Npr1 Ϫ/Ϫ ), heterozygous (Npr1 ϩ/Ϫ ), and wild-type (Npr1 ϩ/ϩ ) mice. This study was performed using newborn (2 days after birth), young (4 weeks of age), and adult (22 weeks of age) Npr1 male mice. The animals were genotyped by PCR analyses of DNA isolated from tail biopsies using primer A (5Ј-GCT CTC TTG TCG CCG AAT CT-3Ј), corresponding to a sequence 5Ј of the mouse Npr1 gene common to both alleles (Npr1 ϩ/ϩ ); primer B (5Ј-TGT CAC CAT GGT CTG ATC GC-3Ј), corresponding to the exon 1 sequence present only in the intact allele (Npr1 ϩ/Ϫ ); and primer C (5Ј-GCT TCC TCG TGC TTT ACG GT-3Ј), corresponding to a sequence in the neomycin resistance cassette present only in the null allele (Npr1 Ϫ/Ϫ ). PCR was carried out in 25 l of reaction mixture containing 50 mM Tris-HCl (pH 8.3), 20 mM ammonium sulfate, 1.5 mM MgCl 2 , 10% Me 2 SO, 100 M dNTPs, 2 units of Taq DNA polymerase, and 40 nM primers. PCR was performed with a 60-s denaturation step at 94°C, a 60-s annealing step at 60°C, and a 60-s extension step at 72°C for 35 cycles using the DNA Thermal Cycler Model 480 as described previously (22). PCR products were resolved on 2% agarose gel. The endogenous band is 500 bp, and the targeted band is 200 bp.
Assessment of Blood Pressure, Heart Rate, and Cardiac Function-Blood pressure and heart rate were measured by a noninvasive computerized tail-cuff method using VisiTech 2000 as described previously (23). Blood pressure and heart rate were calculated as the average of six to seven sessions/day for 6 consecutive days. Cardiac functions of young and adult Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mice were analyzed using two-dimensional echocardiography. Animals were lightly sedated using 0.2 ml of Avertin (Aldrich) and were evaluated using M-mode transthoracic views to measure the left ventricular dimensions, interventricular septal wall thickness, left ventricular posterior wall thickness, and fractional shortening. Digitized M-mode images were obtained using an ultrasound system (Toshiba Power Vision) with a 7-mHz transducer at a sweep speed of 100 mm/s. For each measurement, four consecutive cardiac cycles were traced and averaged.
Assay of Plasma and Ventricular cGMP Levels-Blood samples were collected in tubes containing EDTA and immediately centrifuged at 2500 rpm for 10 min at 4°C. Plasma was separated and stored at Ϫ70°C until used. Frozen ventricular tissue samples were homogenized in 10 volumes of 0.1 M HCl containing 1% Triton X-100. The homogenate was heated at 95°C for 5 min and centrifuged at 600 ϫ g at 22°C, and the supernatant was collected. cGMP levels in plasma and ventricular samples were analyzed using a direct cGMP immunoassay kit (Assay Designs, Inc.) as described previously (24). The results are expressed as picomoles of cGMP/mg of protein.
Northern Blot Analyses of Hypertrophic Marker Genes-Total RNA was isolated from left ventricular tissues of Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mice using TRIzol reagent according to the manufacturer's protocol. To remove genomic DNA contamination, RNA samples were treated with RNase-free DNase I (1 unit/g of RNA) at 37°C for 30 min. The RNA integrity was confirmed by visualization of distinct 28 S and 18 S bands after electrophoresis on 1.5% agarose gel. Total RNA (10 g) was fractionated on 1% formaldehyde-agarose gel and transferred to Hybond nylon membrane (Amersham Biosciences) by capillary action in 10ϫ SSC. Blots were prehybridized in a hybridization solution containing 7% SDS, 0.5 M NaHPO 4 (pH 7.2), and 250 g/ml salmon sperm DNA for 5 h at 65°C and hybridized with [␥-32 P]ATP-labeled oligonucleotide probes for 16 h at 65°C. Blots were washed three times with 2ϫ SSC and 0.2% SDS at room temperature for 30 min and then with 0.5ϫ SSC and 0.2% SDS at 65°C for 30 min before exposure to x-ray film. The sequences of the oligonucleotide probes were as follows: ANP, 5Ј-CCG TABLE I Blood pressure, heart rate, HW/BW ratios, and cardiac structure and function analyses in young and adult Npr1 ϩ/ϩ and Npr1 Ϫ/Ϫ mice Cardiac structure and function analysis was performed, and body weight (BW), blood pressure (BP), heart rate (HR), and HW/BW ratio were measured as described under the "Experimental Procedures." LVEDS, left ventricular end dimension (systolic); LVEDD, left ventricular end dimension (diastolic); IVSTD, interventricular septal wall thickness (diastolic); PWT, posterior ventricular wall thickness (systolic); FS, fractional shortening. Values are expressed as means Ϯ S.E. (n ϭ eight animals in each group).  MHC Protein Isoform Shift Analysis-Myosin was extracted using the method described by Martin et al. (25). Approximately 75 mg of left ventricular tissues from Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mice were minced and washed with ice-cold phosphate-buffered saline (PBS) (pH 7.2). The minced tissues were homogenized in 3 ml of PBS with a Polytron (Brinkmann Instruments) at a setting of 4 (three to four strokes, 30 s each) at 4°C. The homogenate was centrifuged at 1200 ϫ g for 10 min at 4°C. The supernatant was discarded, and the pellet was rewashed with 3 ml of PBS and then recentrifuged at 1200 ϫ g for 10 min. The supernatant was again discarded, and the pellet was dissolved in an extraction solution containing 100 mM Na 4 P 2 O 7 , 5 mM EGTA, and 5 mM dithiothreitol (DTT) (pH 8.6). The homogenate was shaken in an ice bath at 4°C for 60 min, after which it was centrifuged at 12,000 ϫ g for 2 h at 4°C. The supernatant was collected, mixed with equal volume of ice-cold glycerol, and stored at Ϫ20°C. SDS-PAGE was performed essentially as described previously (24). The gels were stained with 0.25% Coomassie Brilliant Blue R-250 solution and destained. The ␣and ␤-MHC protein isoforms were quantified using the Alpha Innotech imaging system.
RNase Protection Assay-RNase protection assay was carried out using the custom-made multiprobe template set for MMP-2, MMP-9, procollagen I, and the GADPH and L32 housekeeping genes. The multiprobe templates were labeled with [␣-32 P]UTP using T7 RNA polymerase following the manufacturer's protocol. Labeled probe (3 ϫ 10 5 cpm) was allowed to hybridize with 20 g of total RNA at 56°C for 16 h. The hybridized mRNA probes were treated with RNase A and extracted with phenol/chloroform. Protected hybrid bands were resolved on 5% denaturing polyacrylamide gel and exposed to radiographic film overnight at Ϫ80°C. Densitometry was performed using the Alpha Innotech imaging system.
MMP Activity Assay-Left ventricular tissues were washed with icecold saline and homogenized with a Polytron in 1:3 (w/v) extraction buffer (pH 5.0) containing 10 mM cacodylic acid, 150 mM NaCl, 20 mM ZnCl 2 , 1.5 mM NaN 3 , and 0.01% Triton X-100 (17). The homogenate was centrifuged at 800 ϫ g for 10 min at 4°C, and the supernatant was collected and concentrated using 30-kDa cutoff microcentrifugal filter devices. The final protein concentrations of myocardial extracts were determined using the Bio-Rad protein assay kit. The extracted samples were then aliquoted and stored at Ϫ20°C until used. MMP activity was measured by an antibody capture method essentially as described by Spinale et al. (26). The spec- ificity and concentration dependence of MMP-2 and MMP-9 activities were initially established using commercially available MMP-2 and MMP-9 standards (1-12 ng/ml). Left ventricular myocardial extracts (25 g of protein) or MMP-2 and MMP-9 standards were incubated overnight at 4°C in a 96-well microtiter plate immobilized with anti-MMP-2 or anti-MMP-9 monoclonal antibody (Amersham Biosciences). After washing the plates with 10 mM Tris-HCl (pH 7.6), a chromogenic peptide substrate solution was added; the reaction was allowed to proceed at 37°C for 1 h; and the absorbance was recorded at 405 nm. The absorbance from the cleaved chromogenic substrate was linear with increasing MMP-2 and MMP-9 activities, with an optimum substrate concentration of 0.4 mM. Assay conditions for MMP-2 and MMP-9 activities were optimized with increasing myocardial protein and substrate concentrations and incubation periods at 37°C. In preliminary studies, MMP-2 and MMP-9 activities were linear with increasing protein concentrations up to 50 g, substrate concentrations up to 1 mM, and incubation periods up to 90 min. The proteolytic activity was reduced by 95% in the presence of the MMP inhibitors 1,10-phenanthroline monohydrate (1 mM) and GM 6001 (0.5 nM; N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide). Actual MMP-2/MMP-9 activities were determined by regression analysis and are expressed as nanograms/h/g of heart tissue. The endogenous levels of MMP-2 and MMP-9 activities were analyzed using the BIOTRACK ELISA activity assay system (Amersham Biosciences). ELISA was performed using the kit according to the manufacturer's protocol. Total MMPs extracted from left ventricular tissues of Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mice were used for enzyme activity assays.
Zymography-To determine the total MMP activities in situ, fresh frozen ventricular tissue sections were incubated with gelatin/Oregon Green (0.5 mg/ml) in zymogram developing buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM CaCl 2 , 1 mM PMSF, and 0.02% NaN 3 at 37°C for 3 h. The slides were washed three times with PBS to remove unbound gelatin. Gelatinase activity resulted in the loss of quenching; therefore, an increase in activity was visualized as a linear increase in fluorescence. MMP inhibitors such as GM 6001 and 1,10-phenanthroline were used to show the specificities of MMP activities. Left ventricular MMP activities were also measured by gelatin zymography. Total MMPs (25 g of protein) extracted from Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mouse hearts were directly loaded onto 10% gel containing 1 mg/ml gelatin under nonreducing conditions (17). Gels were washed twice with 2.5% Triton X-100 (30 min each) and once with substrate buffer (50 mM Tris-HCl, 5 mM CaCl 2 , and 0.02% NaN 3 (pH 7.5)) and then incubated at 37°C for 24 h in fresh substrate buffer. Gels were stained with 0.25% Coomassie Brilliant Blue R-250 and destained until the white lytic bands were visible.
Treatment of Npr1 Mice with an MMP Inhibitor-Young Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mice were treated with the MMP inhibitor GM 6001 and placed randomly into four groups. Groups I and II consisted of Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mice that received subcutaneous injections of PBS and served as controls. Groups III and IV consisted of Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mice that received subcutaneous injections of the MMP inhibitor GM 6001 (100 mg/kg of body weight) twice a week for 4 weeks. The ability of GM 6001 to effectively block MMP activities was tested by incubating a zymogram gel containing ventricular protein extracts and 0.5 nM GM 6001 in the incubation buffer as described above, which completely abolished MMP-2 and MMP-9 activities. At the end of the experimental period, echocardiographic analysis was performed, and cardiac collagen contents, MMP activities, fibrosis, and myocyte cross-sectional areas were measured.
Electrophoretic Mobility Shift Assay-Electrophoretic mobility shift assay was performed as described by Dent et al. (28). A double-stranded oligonucleotide containing the consensus binding site for NF-B was utilized. Oligonucleotides were end-labeled using [␥-32 P]ATP and T4 polynucleotide kinase (New England Biolabs Inc., Beverly, MA) according to the manufacturer's protocol. The binding reaction was initiated by incubating 5 g of nuclear proteins in 5 l of binding buffer (50 mM Tris-HCl (pH 8.0), 750 mM KCl, 2.5 mM EDTA, 0.5% Triton X-100, 62.5% glycerol, and 1 mM DTT) containing 2 g of poly(dI-dC) and radiolabeled oligonucleotide (50,000 cpm) at 22°C for 20 min. Unlabeled competitor assays were performed by adding a 100-fold molar excess of unlabeled NF-B oligonucleotide. The supershift assay was performed with anti-p65 antibody. The DNA-protein complex was resolved from the free labeled DNA by electrophoresis by 5% native PAGE and visualized by autoradiography.
Determination of Hydroxyproline, TNF-␣, and the Collagen Type I/III Ratio-The left ventricular total collagen concentration was quantified from the hydroxyproline content, which was determined by a modified method of Bergman and Loxley (30). The ventricular tissues were homogenized and hydrolyzed in 6 N HCl at 110°C for 24 h in a sealed reaction vial. The hydrolyzed sample was dried using a flash evaporator, and the residue was resuspended in sterile water. The sample was treated with 0.5 ml of chloramine T, vortexed, and left for 5 min. Ehrlich's reagent (3 ml) was added to the sample, and the mixture was vortexed and left for 18 h at room temperature. The intensity of the red coloration that developed was measured at 558 nm. A conversion factor of 8.2 was used to convert hydroxyproline contents to total collagen concentration. Myocardial collagen was extracted using the CNBr method, and the collagen type I/III ratio was determined using the procedure described by Mukherjee and Sen (31). Pro-inflammatory cytokine (TNF-␣) levels in left ventricular tissues from Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mice were analyzed using the ELISA kit following the manufacturer's protocol.
Measurement of Cardiac Hypertrophy and Interstitial Fibrosis-Animals were killed by cervical dislocation, and hearts were isolated. Heart weight and left ventricular weight and their ratio to body weight were calculated as an index of cardiac hypertrophy. Ventricular tissues were fixed in 4% paraformaldehyde solution. Paraffin-embedded tissue sections (5 m) were stained with Masson's trichrome for the presence of interstitial collagen fiber accumulation as a marker of cardiac fibrosis. The ratio of interstitial fibrosis to the total left ventricular area was calculated from 20 randomly selected microscopic fields in five individual sections per heart using ImagePro Plus image analysis software (Media Cybernetics, Inc. Silver Spring, MD).
Antihypertensive Drug Treatments-Experiments were performed using young Npr1 homozygous null mutant (Npr1 Ϫ/Ϫ ) and age-matched wild-type (Npr1 ϩ/ϩ ) mice at 4 weeks of age. All animals were placed in four groups of eight Npr1 Ϫ/Ϫ and eight Npr1 ϩ/ϩ mice (n ϭ 16 animals/ group). Group I mice were kept as a positive control; Group II mice received hydralazine (25 mg/kg/day); Group III mice received captopril (0.5 mg/kg/day); and Group IV mice received bendroflumethiazide (10 mg/kg/day). The drugs were given orally by gavage once a day for 4 weeks. Systolic blood pressure and heart rate were measured before and during the drug treatments for 4 weeks. Animals were killed at the end of the drug treatment periods, and the heart weight/body weight (HW/BW) ratio, cardiac fibrosis, and MMP-9 activity were analyzed.
Statistical Analysis-The results are presented as means Ϯ S.E. Differences between groups were determined using one-way analysis of variance with Dunnett's multiple comparisons post hoc test. A p value Ͻ0.05 was considered significant.

RESULTS
This study was carried out to examine whether permanent ablation of ANP/NPRA signaling activates abnormal cardiac remodeling in newborn, young, and adult Npr1 homozygous null mutant (Npr1 Ϫ/Ϫ ) mice. The data presented in Table I show that adult Npr1 Ϫ/Ϫ mice exhibited 34 Ϯ 7 mm Hg higher systolic blood pressures compared with age-matched wild-type (Npr1 ϩ/ϩ ) mice. Heart rates were significantly decreased (p Ͻ 0.05) in both young and adult Npr1 Ϫ/Ϫ mice compared with age-matched wild-type control mice. The HW/BW ratio in Npr1 Ϫ/Ϫ mice was increased by 52% (global hypertrophy) in young animals at 4 weeks of age, and it further increased to 64% (severe hypertrophy) in adult animals at 22 weeks of age compared with age-matched Npr1 ϩ/ϩ control mice. Myocyte cross-sectional areas were measured in heart sections from both young and adult mutant and wild-type mice and were found to be significantly increased (p Ͻ 0.01) in young Npr1 Ϫ/Ϫ mice at 4 weeks of age and further increased in adult Npr1 Ϫ/Ϫ mice compared with Npr1 ϩ/ϩ mice (Table I). M-mode echocardiographic analysis revealed that young Npr1 Ϫ/Ϫ mice showed increased septal wall thickness and posterior wall thickness and significantly elevated left ventricular end diastolic and systolic dimensions (p Ͻ 0.01) compared with Npr1 ϩ/ϩ mice (Table I). Functional parameters such as fractional shortening and the ejection fraction were significantly reduced in young Npr1 Ϫ/Ϫ mice. Adult null mutant mice showed further increases in septal wall thickness and posterior wall thickness and significantly lower fractional shortening and ejection fractions (p Ͻ 0.001) compared with age-matched wild-type mice, indicating that, in both young and adult Npr1 Ϫ/Ϫ mice, the cardiac function is significantly compromised. M-mode echocardiographic analysis showed progressive cardiac hypertrophy and congestive heart failure in adult Npr1 Ϫ/Ϫ mice. Masson's trichrome staining in left ventricular sections from young and adult Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mouse hearts is shown in Fig. 1  (A-D). Blue staining reflects the intensity of fibrosis in the heart tissues. Increased fibrosis (20%; p Ͻ 0.001) was observed in young Npr1 Ϫ/Ϫ mice, and more pronounced fibrosis (35%; p Ͻ 0.001) was noted in adult Npr1 Ϫ/Ϫ mice compared with age-matched Npr1 ϩ/ϩ counterparts (Fig. 1E). Heart size and left ventricular weight/body weight ratios in young and adult Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mice at 4 and 22 weeks of age are shown in Fig. 1 (F and G). It is evident that Npr1 Ϫ/Ϫ mice had global cardiac hypertrophy at 4 weeks and severe hypertrophy with chamber dilatation at 22 weeks of age. The plasma and ventricular cGMP levels were significantly reduced by 5-and 6-fold in young and adult null mutant mice, respectively, compared with age-matched wild-type mice (Fig. 2).
Electrophoretic mobility shift assay was carried out to ana- lyze NF-B activity in young and adult Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mouse hearts (Fig. 10, A and B). The specificity of the detected NF-B bands was confirmed by competition analyses using a 100-fold molar excess of unlabeled NF-B, which abrogated anti-p65 antibody complex formation and the appearance of the supershift band. Nuclear extracts isolated from young and adult Npr1 Ϫ/Ϫ mice showed significant increases (almost 4-fold) in NF-B binding activity compared with age-matched Npr1 ϩ/ϩ mice. Fig. 10 (C and D) shows the results from Western blot analyses of the NF-B p65 subunit and phospho-IB␣ protein levels in nuclear and cytoplasmic extracts, respectively, from young Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mouse hearts. Significant increases in the p65 (4-fold; p Ͻ 0.001) and phospho-IB␣ (3-fold; p Ͻ 0.001) protein levels were observed in the nuclear and cytoplasmic extracts, respectively, from young Npr1 Ϫ/Ϫ mouse hearts compared with age-matched Npr1 ϩ/ϩ mouse hearts. Furthermore, IB kinase activity increased significantly (4-fold; p Ͻ 0.001) in young Npr1 Ϫ/Ϫ mouse hearts compared with age-matched Npr1 ϩ/ϩ mouse hearts (Fig. 10,  E and F). DISCUSSION The studies presented here demonstrate that Npr1 gene disruption in mice provokes enhanced expression and activation of MMPs and pro-inflammatory cytokines associated with cardiac hypertrophy, fibrosis, and ECM remodeling. The gelatinases (MMP-2 and MMP-9) are considered key enzymes in matrix component degradation and have been suggested to play a critical role in matrix remodeling and left ventricular enlargement (16). Conversely, inhibition of MMPs has been shown to limit ECM destruction and to improve myocardial structure and its function in animal models (15,16,32). Increased expression and activities of MMP-2 and MMP-9 in Npr1 homozygous null mutant (Npr1 Ϫ/Ϫ ) mice are consistent with previously reported observations that MMPs are increased in the failing hearts of both animal models and humans (15,17,33,34). Our present findings are consistent with the notion that an activated ANP/NPRA system can inhibit collagen synthesis (20), which advances the hypothesis that disruption of the NPRA signaling pathway can lead to activation of ECM components and exaggerated cardiac hypertrophy and remodeling. It is expected that an increase in MMP activity would result in a decrease in MMP substrate (collagens). However, the contrary is usually observed: enhanced MMP activity is accompanied by increased fibrosis, whereas attenuated MMP activity is associated with decreased deposition of fibrotic tissues (32,35,36). Indeed, this seems to be paradoxical because the total matrix collagen content is a function of both synthesis and degradation; in turn, the degraded products of matrix proteins can serve as catalysts for collagen synthesis (32,37). As a result, the increased deposition of inappropriately structured fibrotic tissues may occur in the myocardium. Consistent with this notion, the observed parallel increases in total collagen concentrations and MMP levels in Npr1 null mutant mice in this study are a function of both collagen synthesis and degradation. The differences in collagen contents between Npr1 wild-type and null mutant mice are meaningful and significant in both young and adult animals. Our results suggest that increased MMP levels in Npr1 null mutant mouse hearts would be detrimental to the normal collagen matrix; thus, it is not surprising that the changes in the structural properties of collagen seem to be associated with the development of progressive cardiac hypertrophy and ventricular remodeling in Our results also show that expression of MMP-2 and MMP-9 is significantly enhanced and that expression of TIMP-1 and TIMP-2 is attenuated in Npr1 Ϫ/Ϫ mouse hearts. It is known that TIMPs bind to the active site of MMPs, thereby blocking their access to ECM substrates. Previously, it has been shown that TIMP-1 forms a complex with MMP-9 and effectively inhibits its activation (15). Thus, the altered levels of MMPs and TIMPs observed in Npr1 Ϫ/Ϫ mouse hearts could be involved in promoting ventricular remodeling of ECM that may contribute to abnormal cardiomyocyte architecture and organization. Treatment of Npr1 Ϫ/Ϫ mice with the MMP inhibitor GM 6001 attenuated MMP activities and cardiac fibrosis and improved ventricular dilatation, suggesting that activation of MMPs contributes to the remodeling process in mutant mice. To our surprise, MMP inhibitor-treated Npr1 Ϫ/Ϫ mice did not show any significant change in myocyte cross-sectional areas compared with untreated control mice. However, our echocar- diographic analysis demonstrated that MMP inhibitor-treated mutant mice showed attenuated ventricular dilatation and improved fractional shortening. Our findings suggest that MMPs seem to enhance ventricular dilatation and fibrosis, but do not play a direct role in the development of cardiac hypertrophy. Our results further support the previous findings that treatment of rats with spontaneously hypertensive heart failure with the MMP inhibitor PD 166793 prevents cardiac dilatation, preserves contractility, and shows a reduction in myocardial fibrosis compared with untreated rats with spontaneously hypertensive heart failure (38). Furthermore, MMP inhibitors that have been suggested to attenuate the degree of left ventricular dilatation do not have a salutary effect on ventricular hypertrophy (39,40). Nevertheless, several studies suggest that a positive correlation exists between MMP activation and the ventricular remodeling process and that MMP inhibition attenuates both fibrosis and cardiac hypertrophy (15,16,32). Strauss et al. (41) demonstrated that, in balloon-injured arteries, the MMP inhibitor reduces both collagen synthesis and degradation, resulting in reduced accumulation of collagen. These authors suggested that degradation of newly synthesized collagen is an important mechanism regulating collagen accumulation and that MMPs play an integral role in collagen turnover. Indeed, more studies are needed to delineate the exact role of MMPs in the two distinct phenomena of cardiac fibrosis and hypertrophy.
Our results demonstrate that expression of SERCA-2a progressively decreases in hypertrophied left ventricular tissues from Npr1 Ϫ/Ϫ mice. A decrease in SERCA-2a levels has been suggested to contribute to the slowing of relaxation in failing human hearts (42). Thus, altered levels of sarcoplasmic reticulum Ca 2ϩ -handling proteins seem to underlie contractile dysfunction of the heart. Disturbances in diastolic contractile performance are correlated with impaired uptake of Ca 2ϩ into the sarcoplasmic reticulum in animal models and in human dilated cardiomyopathy (43). Thus, the decreased level of SERCA-2a in Npr1 Ϫ/Ϫ mouse hearts observed in our study correlates well with the diastolic dysfunction of these mutant mice. Echocardiographic analysis showed that functional parameters such as fractional shortening and ejection fraction were reduced significantly in both young and adult Npr1 Ϫ/Ϫ mice compared with age-matched Npr1 ϩ/ϩ mice (Table I). These data suggest that Npr1 Ϫ/Ϫ mice experience compromised cardiac function. Our results also provide quantitative assessment of cardiac myosin isoform expression, demonstrating a significant shift from the ␣to ␤-MHC isoform in Npr1 Ϫ/Ϫ mouse hearts. It has been shown that ␣-MHC is detectable in non-failing myocardium and is virtually undetectable in failing and hypertrophied hearts (44). The ␣-MHC isoform contains a high level of ATPase activity, and hearts expressing ␣-MHC have a more rapid contractile velocity compared with hearts expressing ␤-MHC, which is associated with decreased myosin ATPase activity (45). However, the potential functional significance and molecular mechanisms of changes in SERCA-2a gene expression and altered myosin composition in Npr1 Ϫ/Ϫ mice remain to be investigated in more detail.
It should be noted that defects in cardiac relaxation may trigger specific structural and molecular changes in the heart. Recently, it has been suggested that individual cardiac myocytes are significantly larger in Npr1 Ϫ/Ϫ mice at birth and that cardiac hypertrophy predominates independent of blood pressure (46,47). Our results demonstrate that both newborn and young Npr1 null mutant mice display significant increases in left ventricular ␤-MHC mRNAs and a decrease in SERCA-2a mRNA expression, suggesting that hypertrophic genes are probably activated at an early age before blood pressure reaches harmful high levels. To delineate the impact of increased blood pressure on the left ventricular remodeling process, we treated the mutant mice with three different antihypertensive drugs (hydralazine, captopril, and bendroflumethiazide) that have been shown to control hypertension and hypertrophy. To our surprise, normalization of systolic blood pressure did not have any salutary effect in reversing the hypertrophy and fibrosis in Npr1 Ϫ/Ϫ mice, suggesting that NPRA/cGMP signaling has a direct regulatory role in early genes involved in cardiac hypertrophic growth and fibrosis. It has been shown that overexpression of NPRA specifically in cardiomyocytes inhibits the hypertrophic effects of isoproterenol and aortic constriction in mouse models, indicating that NPRA signaling antagonizes cardiac growth in disease states (48). Expression of the ␤-MHC and SERCA-2a genes is considered a sensitive indicator of pathological cardiac hypertrophy but not physiological hypertrophy, and they have been reported to be the principal biomarkers in several models of hypertrophic phenotypes (49,50). Furthermore, increased expression of TGF-␤1 and TNF-␣ is also associated with cardiac hypertro- phy, and blocking the action of these cytokines could prevent myocardial diastolic dysfunction and heart failure (32,51,52). Consistent with these previous findings, our results show that both TGF-␤1 and its receptor are increased, which may contribute to increased myocardial fibrosis and collagen synthesis in mice lacking NPRA. In agreement with our present findings, it has recently been shown that BNP, which activates NPRA, inhibits TGF-␤1-induced ECM protein synthesis in vitro (21). Plasma and ventricular cGMP levels were decreased by almost 5-fold in Npr1 Ϫ/Ϫ mice compared with Npr1 ϩ/ϩ mice. Activation of NPRA by natriuretic peptides increases cGMP production in a number of cell types, and an increased cGMP level is positively correlated with anti-hypertrophic and anti-proliferative effects of natriuretic peptides under in vitro culture conditions (8 -12). Furthermore, the inhibitory action of natriuretic peptides on angiotensin II-induced cardiac hypertrophy is mimicked by 8-bromo-cGMP. The NPRA antagonist HS-142-1 has also been shown to block the anti-hypertrophic effects of ANP by inhibiting the generation of cGMP in neonatal and adult cardiac myocytes (9). Thus, cGMP can be considered as an important mediator of anti-hypertrophic responses to the ANP/NPRA signaling system. Significant increases in NF-B binding activity were observed in both young and adult Npr1 Ϫ/Ϫ mice, suggesting that increased activation of NF-B occurs in the absence of NPRA signaling in mutant mouse hearts. NF-B is a ubiquitous multifunctional signaling system that contributes to cell survival, apoptosis, and inflammation, and it plays a critical role in transcriptional activation of multiple genes that contribute to the development of end organ damage (53). It has been suggested that MMP and pro-inflammatory cytokine genes are regulated by NF-B; however, little is known about the crosstalk between natriuretic peptide and NF-B pathways. The sustained induction of NF-B has been reported in experimental animal and human heart failure conditions (54). The ANP/ NPRA system has been suggested to attenuate production of inflammatory mediators such as TNF-␣ by regulating the NF-B pathway (55). Furthermore, cGMP analogs have been shown to suppress the induction of vascular cell adhesion molecule-1 and expression of hypoxia-associated vascular endothelial growth factor by inhibiting the activation of NF-B (56). Our study shows that NF-B is activated and associated with ventricular remodeling in Npr1 Ϫ/Ϫ mice, indicating a relationship between NF-B activation and myocardial remodeling. Fig. 11 shows a diagrammatic representation of the signaling pathways involved in promoting cardiac hypertrophy and remodeling in the absence of NPRA and cGMP signaling. Our findings show that disruption of the NPRA/cGMP signaling pathway abolishes the local growth-moderating effects and thereby promotes the development of cardiac abnormalities independent of blood pressure. The ANP/NPRA system has been suggested to regulate cardiac function, in particular relaxation; therefore, the disruption of NPRA signaling can lead to impaired cardiac relaxation, resulting in specific structural and molecular changes in Npr1 Ϫ/Ϫ mouse hearts. Nitric oxide, which also utilizes cGMP as a second messenger, has been shown to prevent hypertrophy of neonatal and adult cardiac myocytes (57). In addition, nitric oxide inhibition in ex vivo aortic tissues causes a dose-dependent increase in MMP-9 expression and activity in association with increased NF-B and activator protein-1 activities (58,59). These findings provide strong support for the involvement of the cGMP signaling axis in regulation of the MMPs and the ventricular remodeling process.
In conclusion, several key findings emerge form this study. First, it has provided direct evidence that the MMP-2, MMP-9, and TNF-␣ genes are potently activated in Npr1 Ϫ/Ϫ mouse hearts at an early age and remain increased in adult mutant mice. Second, it has demonstrated that total collagen, the collagen type I/III ratio, and myocardial fibrosis are dramatically increased in adult mutant mouse hearts. Third, it has identified hypertrophic marker genes, including ␤-MHC and TGF-␤1, that are significantly up-regulated in ventricular tissues from both young and adult Npr1 Ϫ/Ϫ mice. Fourth, it has shown a marked shift from the ␣to ␤-MHC protein isoform and a significant decrease in SERCA-2a levels in mutant mouse hearts. Finally, this study has shown enhanced NF-B binding activity and accelerated translocation of the p65 subunit from the cytoplasmic to nuclear fraction of ventricular tissues from Npr1 Ϫ/Ϫ mouse hearts. Together, these findings reveal several features of complex assembly that appear to be unique to cardiac hypertrophy, fibrosis, and remodeling in Npr1 Ϫ/Ϫ mice. FIG. 9. Effect of antihypertensive drug treatments on blood pressure reduction, changes in HW/BW ratios, and MMP-9 activity in young Npr1 ؊/؊ and Npr1 ؉/؉ mice. A-C, blood pressure (BP), HW/BW ratios, and MMP-9 activity, respectively, in young Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mice. Blood pressure was measured before and after drug treatments for 4 weeks. The animals were killed, and HW/BW ratios and MMP-9 activity were analyzed as described under "Experimental Procedures." Animals were left untreated (UNT) or were treated with hydralazine (HDZ; 25 mg/kg/day), bendroflumethiazide (BTZ; 10 mg/kg/day), or captopril (CAP; 0.5 mg/kg/day). Values are expressed as means Ϯ S.E. (n ϭ eight animals in each group). † †, p Ͻ 0.01 (treated Npr1 Ϫ/Ϫ versus treated Npr1 ϩ/ϩ ); ***, p Ͻ 0.001 (untreated Npr1 Ϫ/Ϫ versus treated Npr1 Ϫ/Ϫ ).
FIG. 10. Comparative analysis of NF-B binding and phospho-IB␣ in young and adult Npr1 ؊/؊ and Npr1 ؉/؉ mice. A, autoradiogram of NF-B binding activity in nuclear fractions from Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mice. Unlabeled competitor assays were performed by adding a 100fold molar excess of unlabeled NF-B oligonucleotides. B, densitometric analysis of NF-B binding activity. C, Western blot analysis of p65 and phospho-IB␣ (p-IB␣) proteins in nuclear and cytoplasmic fractions from Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mice. Nuclear and cytoplasmic proteins (20 g/ lane) were separated by 10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and incubated overnight at 4°C with anti-p65 and anti-phospho-IB␣ primary antibodies as described under "Experimental Procedures." D, densitometric analysis of p65 and phospho-IB␣ protein bands. E and F, autoradiogram of IB kinase activity and density units in Npr1 Ϫ/Ϫ and Npr1 ϩ/ϩ mouse hearts. Values are expressed as means Ϯ S.E. (n ϭ eight animals in each group). ***, p Ͻ 0.001 (Npr1 Ϫ/Ϫ versus Npr1 ϩ/ϩ ).
FIG. 11. Diagrammatic representation of NPRA signaling in cardiac hypertrophy and ECM remodeling. Disruption of NPRA signaling leads to impaired cardiac relaxation, which triggers specific structural and molecular changes in Npr1 Ϫ/Ϫ mouse hearts. Activated NF-B translocates into the nucleus and activates MMP and pro-inflammatory cytokine genes. In turn, the absence of NPRA signaling should abolish the local growth inhibitory effects of ANP and BNP, thereby likely promoting the development of cardiac hypertrophy and fibrosis. KHD, kinase homology regulatory domain; GC, guanylyl cyclase catalytic domain; IKK, IB kinase; PKG, protein kinase G.