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.
Atrial (ANP)1 and brain (BNP) natriuretic peptides elicit natriuretic, diuretic, vasorelaxant, and anti-proliferative responses, all of which contribute to the regulation of blood pressure and blood volume homeostasis. 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. 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. 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. In vitro studies have shown that the ANP/NPRA system exerts growth inhibitory effects on hypertrophic agonist-induced proliferation of cardiac myocytes, fibroblasts, and mesangial and human vascular smooth muscle cells. Furthermore, transgenic mice overexpressing ANP have smaller hearts compared with wild-type mice, and ANP gene delivery attenuates cardiac hypertrophy in spontaneously hypertensive rats. 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. 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. 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. Recent studies indicate that abnormal remodeling of myocardial collagen is caused by dysregulation of MMPs and their endogenous inhibitors, TIMPs. 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. 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; however, the mechanism that inhibits collagen production in the heart is not well understood.
The ANP/NPRA system has been implicated as an anti-hypertrophic and anti-fibrotic protective mechanism that moderates the cardiac remodeling process. ANP and BNP have been shown to inhibit fibroblast proliferation, collagen synthesis, and MMP release via the cGMP-dependent pathway and to have a broad functional opposition to transforming growth factor-β1 (TGF-β1)-induced ECM protein synthesis in vitro. 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.
TRIzol reagent was obtained from Invitrogen. Gene-specific primers were purchased from Midland Certified Reagent Co. (Midland, TX). The RETROscript kit was obtained from Ambion Inc. (Austin, TX). Antibodies for MMP-2, MMP-9, TGF-β1, the TGF-β1 receptor (TGF-β1R), and β-actin, and horseradish peroxidase-conjugated IgG secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Crux, CA). The pro-inflammatory cytokine (tumor necrosis factor-α (TNF-α)) enzyme-linked immunosorbent assay (ELISA) kit was obtained from Pierce, and 4′,6-diamidino-2-phenylindole was from Vector Laboratories (Burlingame, CA). The RNase protection assay kit and the custom-made multiprobe set containing MMP-2, MMP-9, procollagen I, glyceraldehyde-3-phosphate dehydrogenase (GADPH), and L32 were from BD Biosciences. The MMP-2 and MMP-9 ELISA activity assay system and [α-32 P]UTP (3000 Ci/mmol) were purchased from Amersham Biosciences. Captopril, hydralazine, and bendroflumethiazide were obtained from Sigma. All other chemicals were reagent-grade.
Npr1 gene-disrupted mice were generated by homologous recombination in embryonic stem cells as described previously. 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-/-), heterozygous (Npr1+/-), and wild-type (Npr1+/+) 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 (5′-GCT CTC TTG TCG CCG AAT CT-3′), corresponding to a sequence 5′ of the mouse Npr1 gene common to both alleles (Npr1+/+); primer B (5′-TGT CAC CAT GGT CTG ATC GC-3′), corresponding to the exon 1 sequence present only in the intact allele (Npr1+/-); and primer C (5′-GCT TCC TCG TGC TTT ACG GT-3′), corresponding to a sequence in the neomycin resistance cassette present only in the null allele (Npr1-/-). PCR was carried out in 25 μl of reaction mixture containing 50 m m Tris-HCl (pH 8.3), 20 m m ammonium sulfate, 1.5 m m MgCl 2, 10% Me 2 SO, 100 μ m dNTPs, 2 units of Taq DNA polymerase, and 40 n m 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. PCR products were resolved on 2% agarose gel. The endogenous band is 500 bp, and the targeted band is 200 bp.
Blood pressure and heart rate were measured by a noninvasive computerized tail-cuff method using VisiTech 2000 as described previously. 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-/- and Npr1+/+ 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.
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 -70 °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 × 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. The results are expressed as picomoles of cGMP/mg of protein.
Total RNA was isolated from left ventricular tissues of Npr1-/- and Npr1+/+ 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/μg 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 μg) was fractionated on 1% formaldehyde-agarose gel and transferred to Hybond nylon membrane (Amersham Biosciences) by capillary action in 10× SSC. Blots were prehybridized in a hybridization solution containing 7% SDS, 0.5 m NaHPO 4 (pH 7.2), and 250 μg/ml salmon sperm DNA for 5 h at 65 °C and hybridized with [γ-32 P]ATP-labeled oligonucleotide probes for 16 h at 65 °C. Blots were washed three times with 2× SSC and 0.2% SDS at room temperature for 30 min and then with 0.5× 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, 5′-CCG GAA GCT GTT GCA GCC TAG GTC CAC TCT GGG CTC CAA TCC TGT CAA TCC TGT CAA TCC TAC CCC CCG AAG CTG GA-3′; BNP, 5′-GTT TAA GCC TCT GGA AAA AGC TAT CTC ACA GGG CCT CTG TTT CTC CTG TAA AGT GGG TTG GGC CAT TCG GA-3′; β-myosin heavy chain (MHC), 5′-GAG GGC TTC ACG GGC ACC CTT AGA GCT GGG AGC ACA AGA TCT ACT CCT CAT TCA TTC AGG CC-3′; and sarco/endoplasmic reticulum Ca 2+-ATPase-2a (SERCA-2a), 5′-TCA GTC ATG CAG AGG CTG GTA GAT GTG TTG CTA ACA ACG CAC ATG CAC GCA CCC GAA CA-3′. The intensity of the bands was quantified using image density analysis software (Alpha Innotech, San Leandro, CA). The expression results of ANP, BNP, β-MHC, and SERCA-2a were normalized to GADPH.
Myosin was extracted using the method described by Martin et al. Approximately 75 mg of left ventricular tissues from Npr1-/- and Npr1+/+ 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 × 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 × g for 10 min. The supernatant was again discarded, and
