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Am J Physiol Heart Circ Physiol 289: H777-H784, 2005. First published March 18, 2005; doi:10.1152/ajpheart.00117.2005
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Cardiac-specific attenuation of natriuretic peptide A receptor activity accentuates adverse cardiac remodeling and mortality in response to pressure overload

Jeetendra B. Patel,1 Maria L. Valencik,2 Allison M. Pritchett,1 John C. Burnett, Jr.,1 John A. McDonald,2 and Margaret M. Redfield1

1Cardiorenal Research Laboratory, Mayo Clinic College of Medicine, Rochester, Minnesota; and 2School of Medicine, University of Nevada, Reno, Nevada

Submitted 4 February 2005 ; accepted in final form 15 March 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Atrial (ANP) and brain (BNP) natriuretic peptides are hormones of myocardial cell origin. These hormones bind to the natriuretic peptide A receptor (NPRA) throughout the body, stimulating cGMP production and playing a key role in blood pressure control. Because NPRA receptors are present on cardiomyocytes, we hypothesized that natriuretic peptides may have direct autocrine or paracrine effects on cardiomyocytes or adjacent cardiac cells. Because both natriuretic peptides and NPRA gene expression are upregulated in states of pressure overload, we speculated that the effects of the natriuretic peptides on cardiac structure and function would be most apparent after pressure overload. To attenuate cardiomyocyte NPRA activity, transgenic mice with cardiac specific expression of a dominant-negative (DN-NPRA) mutation (HCAT D 893A) in the NPRA receptor were created. Cardiac structure and function were assessed (avertin anesthesia) in the absence and presence of pressure overload produced by suprarenal aortic banding. In the absence of pressure overload, basal and BNP-stimulated guanylyl cyclase activity assessed in cardiac membrane fractions was reduced. However, systolic blood pressure, myocardial cGMP, log plasma ANP levels, and ventricular structure and function were similar in wild-type (WT-NPRA) and DN-NPRA mice. In the presence of pressure overload, myocardial cGMP levels were reduced, and ventricular hypertrophy, fibrosis, filling pressures, and mortality were increased in DN-NPRA compared with WT-NPRA mice. In addition to their hormonal effects, endogenous natriuretic peptides exert physiologically relevant autocrine and paracrine effects via cardiomyocyte NPRA receptors to modulate cardiac hypertrophy and fibrosis in response to pressure overload.

natriuretic peptides; diastole; hypertrophy; fibrosis

ATRIAL (ANP) AND BRAIN (BNP) natriuretic peptides (NP) are of cardiomyocyte origin and stimulate production of the intracellular second messenger cGMP via binding to the natriuretic peptide A receptor (NPRA). The natriuretic peptide B receptor (NPRB) preferentially binds C-type NP of endothelial cell origin. A third receptor [natriuretic peptide C receptor (NPRC)] is not linked to guanylyl cyclase and serves as a clearance receptor that, along with digestion by ubiquitous neutral endopeptidases, clears NP from the circulation (27).

Transcripts for all three NP receptors are present in cardiomyocytes and fibroblasts (20), and upregulation of cardiomyocyte NPRA mRNA with hypertrophy has been reported (3). Indeed, in vitro (14, 17, 30, 31, 34, 38, 40) and in vivo (16, 28, 41) studies suggest that the NP and the NP second messenger cGMP have anti-hypertrophic, anti-fibrotic, and prolusitropic effects on the heart. Unfortunately, interpretation of in vivo studies has been confounded by concomitant alterations in load. Furthermore, the density of NPRA receptors on cardiomyocytes is far less than that in lung, kidney, or adrenal tissues (9), raising questions regarding the biological significance of cardiomyocyte NPRA. Holtwick et al. (13) reported that mice with cardiac-specific disruption of the NPRA displayed impaired relaxation and an exaggerated hypertrophic response to pressure overload, suggesting that, indeed, cardiomyocyte NPRA modulates cardiac structure and function independently of load.

The NPRA receptor is composed of extracellular ligand-binding, transmembrane, protein kinase-like, hinge, and catalytic domains (Fig. 1 and Ref. 4 and 5). Receptor homodimerization and ATP binding are required for guanylyl cyclase activity. The region required for homodimer formation resides in the carboxyl terminal portion of the HCAT (for hinge and catalytic) domain (4, 5, 37, 39). A dominant-negative form of the NPRA (DN-NPRA) is based on the requirement of homodimer formation for catalytic activity. Specifically, a truncated form of NPRA containing only the HCAT domain heterodimerizes with wild-type (WT) NPRA and with itself. Cells with coexpression of WT-NPRA and DN-NPRA had low basal cGMP levels that did not increase with ANP stimulation. To abolish residual catalytic activity, Thompson and Garbers (37) performed alanine scanning mutagenesis of the HCAT domain and identified a mutation (D893A) within the cyclase catalytic region that destroyed catalytic activity. Expression of the DN-NPRA mutant subunit resulted in formation of WT- and DN-NPRA receptor complexes that bound ligands normally but were catalytically inactive. The increase of cGMP levels in response to ANP in vitro was inhibited by 85% in COS cells coexpressing WT- and DN-NPRA receptors (37).



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  		Fig. 1. Wild-type (WT) and mutant forms of the natriuretic peptide (NP) A receptor (NPRA). The dominant-negative (DN)-NPRA mutated receptor is truncated and has a much lower molecular mass (~25%) than the full-length WT receptor.

 
To reveal the autocrine or paracrine actions of the NP on cardiomyocytes or adjacent cardiac cells, we created mice with cardiac-specific expression of the DN-NPRA. Our hypothesis was that DN-NPRA mice would demonstrate alterations in cardiac structure and function in the absence of alterations in blood pressure. Additionally, because pressure overload-induced left ventricular (LV) hypertrophy is associated with enhanced production of NP, increases in cardiac NPRA receptor transcripts, and reduced NPRC transcripts (3), we speculated that the autocrine or paracrine effects of the NP may be most apparent in states of pressure overload.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All experimental procedures were designed in accordance with National Institutes of Health guidelines and approved by the Mayo Foundation, University of Utah, and Salt Lake City Veterans Administration Medical Center Institutional Animal Care and Use Committees. Transgenic animals were created in FVB/Ntac mice (Taconic Farms, Germantown, NY). Only male mice were studied. Nonbanded mice were studied at 8–12 wk of age. Suprarenal aortic banding was performed at 8–12 wk of age with groups of mice studied at 11 and at 21 days after banding.

Cardiac-specific expression of DN-NPRA. A transgenic construct containing a FLAG-tagged HCAT subunit of the NPRA receptor cDNA (HCAT-D893A; see Ref. 37) under the control of cardiac-specific mouse {alpha}-myosin heavy chain ({alpha}-MHC) promoter (33) was isolated, purified, and injected in the pronuclei of FVB/N zygotes that were then surgically implanted into pseudopregnant Swiss/CD1 females. To genotype the progeny, DNA was extracted using the Qiagen Dneasy Tissue kit (Valencia, CA) or the Isoquick Nucleic Acid Extraction kit (Orca Research, Bothell, WA). PCR was performed to determine transgene integration. Primers were specific to the NPRA coding region (5'-forward primer: NPR 2651–2670: 5'-CCT GGA CAA CCT GCT GTC AC-3' and 3'-reverse primer: NPR 3151–3131: 5'-GAC CTG TGT GGA TGC CAA TG-3'). To confirm single integration of the transgene, Southern blot analysis of genomic DNA prepared from tail biopsies was performed. Western analysis was performed in heart tissue extracts from offspring to determine the level of mutant protein expression.

Immunoblotting. Hearts were homogenized in 50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.5% Triton X-100 and centrifuged (16,000 g, 15 min, 4°C), and supernate protein concentration was determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL). For immunoblotting, equal amounts of protein (1–10 µg) were electrophoresed in 2x SDS reducing buffer on an 8–16% gradient SDS-PAGE gel (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose (Hybond-N; Amersham). Two separate immunoblots were performed. First, antibody M5 (Sigma, St. Louis, MO) was used to detect FLAG-tagged HCAT-D893A in different lines of DN-NPRA mice. To determine uniformity of DN-NPRA expression, immunohistochemical analysis of heart sections was performed.

Effective ablation of NPRA activity in the DN-NPRA model requires high expression of the DN-NPRA relative to the WT-NPRA. In a second experiment, the relative amount of WT- and DN-NPRA in cardiomyocytes was assessed using an antibody (a generous gift from Dr. David Garbers) that recognizes the carboxyl terminus of both the WT-NPRA and DN-NPRA proteins (37). Because the DN-NPRA mutation is a much truncated form of the NPRA, it migrates to the {approx}30 kDa location on the blot, whereas the WT-NPRA migrates to 119 kDa.

Blood pressure. After training was completed, systolic blood pressure was measured (10 measurements over 30 min) one time daily for 5 days in conscious nonbanded mice (Visitech System, Apex, NC), and the average daily values for the 5 days were averaged.

Suprarenal aortic banding as a model of pressure overload. Mice were anesthetized with Avertin [0.015 ml/g body wt ip, 2,2,2-tribromoethanol; Fluka Chemika] with supplemental dosing (0.1 ml) as needed. Via a left-sided flank incision, the suprarenal abdominal aorta was banded with 6-0 silk suture tied over a 29-gauge needle that was then removed (36). In a subgroup of banded and nonbanded conscious WT mice, proximal aortic pressure was measured 3 wk after banding to confirm that this intervention produced pressure overload. A 1.4-Fr manometer-tipped catheter (Millar Instruments, Houston, TX) was secured in the aorta via the right carotid artery under avertin anesthesia. After recovery from anesthesia, pressure was measured in these conscious, spontaneously moving mice.

Echocardiography. Avertin-anesthetized mice underwent two-dimensional targeted M-mode echocardiography (GE System 5; GE, Horten, Norway) with a 10 MHz probe. LV end-diastolic and end-systolic dimensions and septal and posterior diastolic wall thickness were measured off-line. Fractional shortening and LV mass were calculated as previously described (11).

Catheterization and hemodynamic analysis. After echocardiography, a manometer-tipped catheter was inserted in the LV via the right carotid artery. Parameters obtained or derived included LV peak systolic and end-diastolic pressure, maximal rates of pressure change (maximal and minimal dP/dt), and the time constant of isovolumic relaxation calculated using the logistic method (Sonolab, Sonometrics, London, Ontario; see Ref. 26).

Tissue and blood harvest. After catheterization, blood was collected from the carotid artery, and the heart was removed for total cardiac and LV weight. Sections of the LV were flash-frozen in liquid nitrogen or preserved in formalin for histological analysis (picrosirus red). The degree of perivascular and interstitial fibrosis was assessed using computer-assisted histomorphometry (Ziess Vision, Hallbergmoos, Germany). Plasma ANP concentration was measured without extraction by RIA as previously described (2).

Myocardial cGMP and guanylyl cyclase activity. In nonbanded (DN-NPRA, n = 11 and WT-NPRA, n = 11) and banded (DN-NPRA, n = 10 and WT-NPRA, n = 11) mice, LV samples were harvested and flash-frozen for measurement of myocardial cGMP concentrations. Cardiac tissue was homogenized, and, after extraction, cGMP was measured by RIA as previously described (12). Results were standardized per milligram protein, as measured by the Lowry method described previously (12). Guanylyl cyclase activity (basal and BNP stimulated) was measured in membranes isolated from whole hearts (DN-NPRA, n = 4 and WT-NPRA, n = 4 mice) using methods previously described (9). The membrane fractions were divided into four aliquots. From these aliquots, we measured protein concentration, background cGMP level, cGMP levels after 20 min of incubation (to calculate basal guanylyl cyclase activity), and cGMP levels after incubation with BNP (10–4 M) for 20 min (to calculate BNP-stimulated guanylyl cyclase activity). Activity was expressed as picomoles cGMP per milligram protein per 20 min.

Statistical analysis. Data are expressed as means ± SD. Comparison between groups was performed using an unpaired Student's t-test. A P value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cardiac-specific transgene expression. Initial PCR screening identified 10 viable potential founders that had integrated the DN-NPRA. When mated with WT FVB/N mice, five of the potential founder animals transmitted the transgene to progeny yielding the expected Mendelian ratio. Southern blot analysis of these lines revealed a single transgene integration site. Immunoblot analysis confirmed robust expression of the DN-NPRA mutation in ventricular tissue (Fig. 2A). The transgenic line with uniform expression of DN-NPRA as determined by immunohistochemistry was expanded for subsequent analysis. Immunoblot analysis with antibody AO34, which recognizes an epitope common to both proteins, demonstrated robust expression of DN-NPRA relative to endogenous NPRA, which was not detected by immunoblot analysis (Fig. 2B). Thus the ratio of DN-NPRA to WT receptor in the cardiomyocyte is extremely high.



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  		Fig. 2. Expression of DN-NPRA transcript and protein in mouse hearts. A: immunoblotting in hearts from 3 separate lines of transgenic DN-NPRA mice demonstrating robust expression of the mutant NPRA. B: immunoblotting in WT-NPRA and DN-NPRA transgenic mice using only the antibody that recognizes the COOH-terminus of both the WT- and DN-NPRA proteins. There is dense staining of the mutant DN-NPRA protein at {approx}30 kDa in the transgenic DN-NPRA mice but no staining for the DN-NPRA in the WT mouse or in the rat. There is no detectable staining at 119 kDa, where the WT receptor should migrate in any animal, indicating that the amount of NPRA expression in mouse hearts is below the level detectable by immunoblotting with this antibody.

 
Effect of cardiac-specific DN-NPRA expression on guanylyl cyclase activity, myocardial cGMP levels, and plasma ANP levels. In whole heart membrane fractions, guanylyl cyclase activity was reduced in DN-NPRA vs. WT-NPRA hearts (Fig. 3A). Furthermore, BNP enhanced guanylyl cyclase activity in WT-NPRA but not DN-NPRA hearts (Fig. 3A). Myocardial cGMP levels were similar in nonbanded DN-NPRA and WT-NPRA mice. Myocardial cGMP levels were increased in banded WT-NPRA compared with nonbanded WT-NPRA mice but were similar in banded DN-NPRA compared with nonbanded DN-NPRA mice. Myocardial cGMP levels were reduced in banded DN-NPRA compared with banded WT-NPRA mice (Fig. 3B). Log plasma ANP concentrations were similar in DN-NPRA and WT-NPRA mice in both the unstressed and banded states (Fig. 3C). Log plasma ANP concentration was increased in banded compared with nonbanded WT-NPRA mice and tended to be increased in banded compared with nonbanded DN-NPRA mice. Although the normal values for plasma ANP levels in the normal mouse vary widely (30–210 pg/ml; see Refs. 1, 6–8, 13, 16, 24), plasma ANP values in nonbanded WT-NPRA mice (mean value 186 pg/ml) were within the previously reported range.



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  		Fig. 3. Guanylyl cyclase activity, myocardial cGMP concentrations, and log plasma atrial natriuretic peptide (ANP) concentrations in WT-NPRA and DN-NPRA mice. All data shown as means ± SD. A: in nonbanded mice, basal and brain natriuretic peptide (BNP)-stimulated guanylyl cyclase activity in whole heart membrane fractions was reduced in DN-NPRA (n = 4) compared with WT-NPRA (n = 4) mice. Enzymatic activity is expressed as picomoles of cGMP formed per milligram protein over 20 min. B: myocardial cGMP concentration was similar in hearts harvested from nonbanded DN-NPRA (n = 11) and WT-NPRA (n = 11) mice. Myocardial cGMP concentration was reduced in banded DN-NPRA (n = 10) mice compared with banded WT-NPRA (n = 11) mice. Myocardial cGMP concentration increased in response to banding in WT-NPRA mice but not in DN-NPRA mice. C: log plasma ANP concentration was similar in nonbanded DN-NPRA (n = 13) and WT-NPRA (n = 15) mice and in banded DN-NPRA (n = 28) compared with banded DN-NPRA (n = 32) mice. Log plasma ANP concentration was higher in banded compared with nonbanded WT-NPRA mice and tended to be higher in banded compared with nonbanded DN-NPRA mice.

 
Effect of cardiac-specific DN-NPRA expression on blood pressure. Conscious systolic blood pressure and conscious heart rate were not different in DN-NPRA vs. WT-NPRA mice (Table 1).


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  		Table 1. Cardiac structure and function in DN-NPRA and WT-NPRA littermate control mice in the absence of aortic banding

 
Effect of cardiac-specific DN-NPRA expression on LV structure and function in unbanded mice. In general, cardiac-specific expression of the DN-NPRA did not alter LV structure or function relative to WT-NPRA mice in the absence of pressure overload (Table 1). However, there was a modest (but statistically significant) increase in fractional shortening observed in the DN-NPRA mice.

Suprarenal aortic banding as a model of pressure overload. Central aortic pressure was higher in banded (140 ± 19 mmHg/95 ± 15 mmHg, n = 13) compared with nonbanded WT-NPRA (125 ± 10 mmHg/86 ± 10 mmHg, n = 15, P < 0.05) mice, confirming that suprarenal aortic banding produced pressure overload.

Response to suprarenal aortic banding in DN-NPRA mice and WT-NPRA mice. Procedural and periprocedural (death within ≤3 days of banding) mortality was similar in DN-NPRA and WT-NPRA mice. In contrast, late (death >3 days after banding) mortality was increased in DN-NPRA mice compared with banded WT-NPRA mice (Fig. 4). Given the increased mortality, we speculated that DN-NPRA mice surviving to 21 days might not be representative of the entire group. Accordingly, we assessed LV structure and function in a subgroup of banded DN-NPRA and banded WT-NPRA mice at 11 days postbanding.



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  		Fig. 4. Survival after suprarenal aortic banding in WT-NPRA and DN-NPRA mice. Survival after suprarenal aortic banding is reduced in DN-NPRA compared with WT-NPRA mice.

 
At echocardiography, banded DN-NPRA mice had increased calculated LV mass/body weight compared with banded WT-NPRA (Fig. 5A). This finding was confirmed at autopsy where the heart/body weight (Fig. 5B) and the LV/body weight (Fig. 5C) were higher in banded DN-NPRA mice compared with banded WT-NPRA mice. Banded DN-NPRA mice had increased perivascular, interstitial, and overall LV fibrosis compared with banded WT-NPRA. Because the difference in fibrosis was similar (and statistically significant) in mice studied at 11 or 21 days after banding, group data in all banded mice are shown (Fig. 6). Cardiac catheterization demonstrated that heart rate was similar in banded DN-NPRA (456 ± 15 beats/min, n = 22) and WT-NPRA (492 ± 74 beats/min, n = 24, P = 0.10) mice. Peak LV systolic pressure was also similar in banded DN-NPRA (92 ± 13 mmHg) vs. WT-NPRA (92 ± 19 mmHg, P = 0.97) mice. However, banded DN-NPRA mice had increased tau (slower relaxation) and higher LV end-diastolic pressure compared with banded WT-NPRA (Fig. 7). These abnormalities tended to be more apparent in mice studied 21 days after banding. Although peak +dP/dt tended to be lower in all banded DN-NPRA (5,754 ± 1,481 mmHg/s) compared with banded WT-NPRA mice (6,362 ± 1,680 mmHg/s), this difference was not significant (P = 0.20), and no significant difference in fractional shortening was detected between the two groups.



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  		Fig. 5. Echocardiographic and autopsy assessment of hypertrophy in banded DN-NPRA compared with WT-NPRA mice. Data shown as means ± SD. When normalized to body weight, echocardiographic estimation of left ventricular (LV) mass is increased in DN-NPRA compared with WT-NPRA mice (A). At autopsy, heart weight (B) and LV weight (C) normalized to body weight were increased in banded DN-NPRA mice. Data are shown for mice studied at 11 (11 day; 15 WT-NPRA and 15 DN-NPRA mice) or 21 (21 day; 20 WT-NPRA and 15 DN-NPRA mice) days after banding and for all banded mice (All).

 


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  		Fig. 6. Interstitial, perivascular, and overall collagen volume fraction is increased in banded DN-NPRA compared with banded WT-NPRA mice. Data shown as means ± SD. Hearts from DN-NPRA mice (n = 27) displayed increased overall (top left), perivascular (top middle), and interstitial (top right) fibrosis compared with banded WT-NPRA mice (n = 25). Grouped data from mice studied at 11 or 21 days after banding are shown. Representative examples of LV sections stained with picrosirus red from WT-NPRA (bottom left) and DN-NPRA (bottom right) are shown.

 


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  		Fig. 7. Diastolic function is impaired in banded DN-NPRA compared with banded WT-NPRA mice. The time constant of isovolumic relaxation (tau, ms) is increased (left) in banded DN-NPRA vs. WT-NPRA mice at 21 days and tended to be increased when all banded DN-NPRA mice are considered. LV end-diastolic pressures are higher (right) in banded DN-NPRA mice at 21 days and when all banded mice are considered. Nos. of mice studied in each group were 9 WT-NPRA and 10 DN-NPRA mice at 11 days and 13 WT-NPRA mice and 12 DN-NPRA mice at 21 days.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Cardiomyocyte-specific disruption of NPRA activity was devoid of effects on systemic blood pressure but resulted in enhanced hypertrophic and fibrotic remodeling, impaired diastolic function, and increased mortality in response to pressure overload. Use of cardiac-specific disruption of NPRA activity reveals the direct physiological effects of endogenous NP on cardiac structure and function independent of secondary effects related to their potent load-reducing actions. A single other study using cardiomyocyte-specific NPRA ablation also reported enhanced hypertrophic remodeling and diastolic dysfunction in mutant mice. The current data, using a different strategy for cardiomyocyte NPRA disruption and an alternative and more prolonged pressure overload model, independently confirm the direct anti-hypertrophic effects of the endogenous NP and importantly extend the previous study by demonstrating marked accentuation of fibrotic remodeling and increased mortality in banded DN-NPRA mice subjected to pressure overload. These data serve to establish the physiological relevance of the NP system as an important autocrine paracrine regulator of cardiac structure and function that is protective, both in terms of preventing LV remodeling and mortality in states of pressure overload.

Transcripts for NPRA, NPRB, and NPRC are present in isolated cardiomyocytes and fibroblasts (20). NPRA mRNA is upregulated, whereas NPRC is downregulated, in response to pressure overload (3). Although these studies suggest the existence of NPRA in cardiomyocytes and fibroblasts, the level of NPRA protein present in cardiomyocytes in vivo and its functional significance has been difficult to establish. Using sensitive and specific antibodies, Goy et al. (9) were unable to detect NPRA protein in cardiac tissue by immunoblotting. Nevertheless, the study of Goy et al. and our findings confirm measurable guanylyl cyclase activity in whole heart membrane preparations. Although such preparations may include membranes from noncardiomyocyte cells, we found that guanylyl cyclase activity was reduced in the DN-NPRA hearts and that the BNP stimulated increases in guanylyl cyclase activity we observed in WT-NPRA hearts were absent in DN-NPRA hearts. These findings are consistent with cardiomyocyte NPRA content and activity and confirm that the DN-NPRA mutation attenuates cardiomyocyte NPRA activity. Our findings are further supported by the work of Holtwick et al. (13) in which a cardiac-specific NPRA knockout similarly possessed reduced guanylyl cyclase activity in whole heart membrane preparations. The augmentation in guanylyl cyclase activity in response to BNP in the WT-NPRA mice was quite modest (9.6%). This modest stimulation may make it difficult to demonstrate stimulation of noncardiomyoyte guanylyl cyclase activity in the setting of elimination of the cardiomyocyte component. Of note, Goy et al. (9) found no significant increase in guanylyl cyclase activity in membrane fractions of hearts from WT mice with BNP (but did see a modest response with ANP). Although BNP is recognized to be a less potent stimulator of NPRA than ANP, we had chosen BNP for its clinical relevance, since therapeutic strategies with the NP have focused on BNP.

Although guanylate cyclase activity was reduced in nonbanded DN-NPRA and although myocardial cGMP levels were reduced in banded DN-NPRA mice, we did not find reduced myocardial cGMP levels nor altered structure or function in nonbanded DN-NPRA mice. We speculate that soluable guanylyl cyclase and noncardiomyocyte particulate guanylyl cyclase may contribute most of the basal myocardial cGMP, with cardiomyocyte particulate guanylyl cyclase playing a larger role in hypertrophy when both NP production and cardiomyocyte NPRA expression are upregulated (3).

In cultured myocytes, the NP inhibit (31, 32, 34, 40) and NPRA receptor antagonists (14) potentiate the hypertrophic response to humoral stimulators of cardiomyocyte growth. Although murine models with systemic NPRA ablation (18, 25, 28) or systemic overexpression of the ANP (1) display altered cardiac growth, potent effects on loading conditions in these models complicate interpretation. Indeed, it is important to consider the interaction between load and humoral regulation of cardiac growth in vivo. In vitro studies in cardiomyocytes have consistently demonstrated that the NP and factors such as ANG II and endothelin modulate cardiomyocyte growth. In apparent contrast, in vivo studies have shown that pressure overload hypertrophy proceeds unabated in mice with systemic ablation of the ANG II receptor and that cardiac atrophy occurs in response to chronically reduced load despite marked activation of ANG II and endothelin (10, 22). Such studies suggest that, in vivo, load may be the principal determinant of cardiomyocyte growth. In contrast, cardiac-specific attenuation of NPRA activity, whether via an overexpression of a DN-NPRA mutation or NPRA ablation (13), accentuates hypertrophy independent of load, confirming that the endogenous NP system modulates the interaction of load and cardiomyocyte growth.

Although attenuation of cardiomyocyte NPRA with the DN-NPRA transgene showed a modest accentuation of the hypertrophic response to pressure overload, the novel findings here were the dramatic effects on the fibrotic response to pressure overload and the increased mortality observed in banded mutant mice. These findings confirm and extend those of Holtwick et al. (13), who examined only interstitial fibrosis 10 days after transverse aortic banding and found mildly increased fibrosis in banded mice with cardiac-specific ablation of the NPRA. Furthermore, this study did not report an effect on mortality during the 10-day postbanding observation period. Because male mice with ablation of the BNP gene exhibit a more pronounced fibrotic response and higher mortality than females (36), the exclusive use of males, the model of pressure overload, and the time course examined in the current study may have influenced the more robust fibrotic phenotype and the increased mortality observed.

Although NPRA receptors are present on fibroblasts and exert anti-fibrotic effects (17, 20, 38), the use of the {alpha}-MHC promoter precludes effects on fibroblast NPRA in the current model. Thus increased fibrosis with attenuation of cardiomyocyte NPRA is most likely mediated by an effect on cardiomyocyte production of pro- or anti-fibrotic cytokines. Reduction in cardiomyocyte production of anti-fibrotic cytokines with iron overload is thought to contribute to cardiac fibrosis in hemochromotosis (23). Studies in cultured cardiomyocytes demonstrate that NP reduce aldosterone synthase gene expression (15). Mice with systemic ablation of BNP demonstrate cardiac fibrosis associated with upregulation of cardiac angiotensin-converting enzyme and transforming growth factor-{beta} (TGF-{beta}) in the absence of altered blood pressure (36). Cardiomyocytes produce ANG II (19) and TGF-{beta} (29), and these and other profibrotic and proinflammatory cytokines of cardiomyocyte origin may be regulated by NP via the cardiomyocyte NPRA. Thus our findings support the importance of cardiomyocyte-fibroblast interaction in the regulation of cardiac structure.

Although mice with systemic ablation of the NPRA (28) displayed hypertension and increased mortality, we found increased mortality in banded DN-NPRA mice in the absence of systemic hypertension. These data support a cardioprotective role for the NP independent of their effects on load. The absence of apparent heart failure or hypertension and the significant increase in myocardial fibrosis suggest an arrhythmic basis for the increased mortality, but further studies are needed to define the electrophysiological phenotype.

The cardiac-specific NPRA null mice used in the study of Holtwick et al. (13) displayed systemic hypotension and increased plasma levels of ANP, effects not observed in the DN-NPRA mice. Holtwick et al. postulated that the unexpected hypotension was related to the higher ANP levels and suggested two mechanisms for the higher ANP levels (increased ANP production related to enhanced hypertrophy observed in the cardiomyocyte-specific NPRA null mice or abolition of an autoregulatory mechanism by which ANP might inhibit its own secretion via the cardiomyocyte NPRA). In these regards, it is of note that, in mice with "global" ablation of the NPRA as reported by two separate laboratories, plasma levels of ANP were not elevated. Lopez et al. (25) found no effect on ANP levels despite the presence of hypertension in the systemic NPRA knockout, and Ellmers et al. (8) found an increase in ANP in males but not females with their systemic NPRA knockout. In these mice, not only would there be a lack of any negative feedback effect, since cardiomyocyte and systemic NPRA would be ablated, but there is also a much more significant degree of hypertension and hypertrophy than noted in the cardiac-specific NPRA null mice, which would promote increased ANP production by the ventricle. Thus, given the conflicting findings in the systemic and cardiac-specific null mice, as well as our own data, we can only conclude that the increase in ANP with NPRA ablation is inconsistently observed. We did observe a modest increase in ANP levels in response to banding in both groups (WT-NPRA and DN-NPRA), suggesting that our assay is valid and sensitive to altered ANP levels, but did not observe an effect of the DN-NPRA mutation on the plasma ANP levels in the nonbanded state where there was no LV hypertrophy (LVH) nor in the banded state where the DN-NPRA mice had more LVH than WT mice. The augmentation of LVH in the DN-NPRA mice (and in the cardiomyoctye-specific NPRA null mice in the Holtwick study) is relatively modest. Indeed, when measuring LV mass by echocardiography, a measurement with considerably more variability than autopsy-derived LV mass, the difference between the two strains was less dramatic. Thus differences in ANP resulting from differences in LVH may not result in demonstrable differences in ANP levels in banded mice. If increases in ANP are totally because of a loss of NPRA-mediated negative feedback regulation of ANP secretion, the absence of increased ANP levels or hypotension in the DN-NPRA model may suggest a less complete inhibition of cardiomyocyte NPRA function.

In conclusion, the current study defines the physiological significance of cardiac-specific NP actions. A cardioprotective role for the cardiomyocyte NPRA to limit the hypertrophic and fibrotic response to pressure overload, thus reducing mortality, is suggested. These findings may have therapeutic implications. Indeed, a recent study demonstrated that augmentation of the NP second messenger cGMP via chronic inhibition of cGMP-specific phosphodiesterase (PDE5A) attenuates load-induced hypertrophy, fibrosis, and myocardial dysfunction independent of changes in load (35). Together, these studies support a novel extension of the neurohumoral hypothesis whereby augmentation of endogenous anti-hypertrophic and anti-fibrotic factors represents an alternative to blockade of prohypertrophic, profibrotic pathways or reduction of load alone.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by the Marriott Foundation, the Miami Heart Research Institute, the Mayo-Dubai Healthcare City Research Program, and National Heart, Lung, and Blood Institute Grants T32-HL-07111–27 and HL-64112.


   ACKNOWLEDGMENTS
 
We are indebted to Gerald Harders, Chris Hunter, and Sharon Sandberg for expert technical assistance.

Current address for A. M. Pritchett: Baylor College of Medicine, Department of Medicine-Cardiology, Mail Stop BCM285, One Baylor Plaza, Houston, TX 77030.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. M. Redfield, Cardiorenal Research Laboratory, Guggenheim 9, Mayo Clinic, 200 First St., Southwest, Rochester, MN 55905 (e-mail: redfield.margaret{at}mayo.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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 METHODS
 RESULTS
 DISCUSSION
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