Cardiac hypertrophy in transgenic rats expressing ... - Semantic Scholar

Cardiac hypertrophy in transgenic rats expressing a dominant-negative mutant of the natriuretic peptide receptor B Thomas H. Langenickel*†‡§, Jens Buttgereit*‡¶储, Ines Pagel-Langenickel†¶, Maren Lindner*, Jan Monti¶, Knut Beuerlein**, Nidal Al-Saadi¶, Ralph Plehm*, Elena Popova*, Jens Tank¶, Rainer Dietz¶, Roland Willenbrock¶††, and Michael Bader*‡‡ *Max Delbru¨ck Center for Molecular Medicine, Berlin-Buch, Robert-Ro¨ssle-Strasse 10, D-13092 Berlin, Germany; ¶Franz Volhard Clinic, Humboldt University, Charite´ Campus Berlin-Buch, D-13125 Berlin, Germany; 储Department of Biology, Chemistry, and Pharmacy, Free University of Berlin, D-14195 Berlin-Dahlem, Germany; and **Rudolf Buchheim Institute for Pharmacology, University Clinics, D-35392 Giessen, Germany Edited by David L. Garbers, University of Texas Southwestern Medical Center, Dallas, TX, and approved January 13, 2006 (received for review November 18, 2005)

C-type natriuretic peptide 兩 knockdown

T

he family of natriuretic peptides (NP) consists of the structurally homologous but genetically distinct peptide hormones atrial NP (ANP), brain NP (BNP), and C-type NP (CNP). ANP and BNP are predominantly synthesized in atrial and ventricular cardiomyocytes (CMC) and released upon stretch into the circulation (1). CNP is expressed in a wide variety of tissues and acts locally as an autocrine and paracrine hormone (2). The NP exert their actions by activating guanylyl cyclasecoupled cell surface receptors. ANP and BNP bind specifically to NP receptor (NPR) A, whereas CNP activates NPR-B. ANP, BNP, and CNP display comparable affinity to NPR-C, which lacks intrinsic guanylyl cyclase activity and acts in part as a clearance receptor (3, 4). NP regulate a variety of physiological processes by intracellular accumulation of their second messenger cGMP (cGMP). The accumulation of cGMP upon ligand stimulation requires a dimerization of receptor molecules including the cytoplasmatic kinase homology domain. This process leads to a tight contact of two guanylyl cyclase domains resulting in conversion of Mg2⫹-GTP to 3⬘,5⬘-cGMP (5, 6). ANP, BNP, and their receptor, NPR-A, have been established as regulators of blood pressure, water and electrolyte homeostasis, and cellular growth. In vitro studies demonstrated inhibitory effects on www.pnas.org兾cgi兾doi兾10.1073兾pnas.0510019103

proliferation of vascular smooth muscle cells and CMCs as well as extracellular matrix secretion of cardiac fibroblasts (7–9). Genetic models revealed insight into the complementary role of these NP with respect to regulation of myocardial structure. Disruption of the murine NPR-A gene resulted in salt-resistant hypertension, cardiac hypertrophy, and fibrosis (10–12). Gene targeting of ANP led to salt-sensitive hypertension and cardiac hypertrophy, whereas BNP knockout mice exhibited a marked cardiac fibrosis without hypertension or hypertrophy (13–15). In contrast to ANP and BNP, CNP does not exert systemic effects, but rather acts locally. In vitro studies have shown that CNP mediates antiproliferative and antihypertrophic actions in neonatal CMCs, vascular smooth muscle cells, and cardiac fibroblasts (8, 16, 17). It has not been resolved whether these actions were strictly mediated by NPR-B or whether crossactivation of NPR-A was involved. Targeted disruption of the CNP gene in genetically modified mice resulted in dwarfism due to impaired longitudinal growth of long bones (18). The disruption of the NPR-B gene resulted in impaired endochondral ossification and developmental defects of the female reproductive organs. Blood pressure measurements performed on NPR-B-null mice revealed no differences between knockout and wild-type littermates (19). However, these genetically modified mice died prematurely because of severe skeletal malformation and could not be subjected to further cardiovascular phenotyping. Thus, the currently available genetic models are not suitable to fully characterize the function of NPR-B in the cardiovascular system. We therefore developed a model that allowed a functional down-regulation of the NPR-B signaling by overexpressing a dominant-negative mutant in transgenic rats. These rats are viable, have a normal lifespan, and are accessible to complex cardiovascular phenotyping, thereby overcoming the limits related to a complete deletion of NPR-B or CNP. Our model presented in this study provides new insights in the physiology of NPR-B and evidence that NPR-B is involved in regulation of CMC growth. Conflict of interest statement: No conflicts declared. This paper was submitted directly (Track II) to the PNAS office. Abbreviations: NP, natriuretic peptide; CNP, C-type NP; ANP, atrial NP; BNP, brain NP; NPR, NP receptor; CMC, cardiomyocyte; AVP, Arg-vasopressin. †Present

address: National Heart, Lung, and Blood Institute, 50 Center Drive, Building 50兾4529, Bethesda, MD 20892.

‡T.H.L. §To

and J.B. contributed equally to this work.

whom correspondence may be sent at the † address. E-mail: [email protected].

††Present ‡‡To

address: St. Elisabeth Hospital, D-06110 Halle, Germany.

whom correspondence may be addressed. E-mail: [email protected].

© 2006 by The National Academy of Sciences of the USA

PNAS 兩 March 21, 2006 兩 vol. 103 兩 no. 12 兩 4735– 4740

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Natriuretic peptides (NP) mediate their effects by activating membrane-bound guanylyl cyclase-coupled receptors A (NPR-A) or B (NPR-B). Whereas the pathophysiological role of NPR-A has been widely studied, only limited knowledge on the cardiovascular function of NPR-B is available. In vitro studies suggest antiproliferative and antihypertrophic actions of the NPR-B ligand C-type NP (CNP). Because of the lack of a specific pharmacological inhibitor, these effects could not clearly be attributed to impaired NPR-B signaling. Recently, gene deletion revealed a predominant role of NPR-B in endochondral ossification and development of female reproductive organs. However, morphological abnormalities and premature death of NPR-B-deficient mice preclude detailed cardiovascular phenotyping. In the present study, a dominant-negative mutant (NPR-B⌬KC) was used to characterize CNP-dependent NPR-B signaling in vitro and in transgenic rats. Here we demonstrate that reduced CNP- but not atrial NP-dependent cGMP response attenuates antihypertrophic potency of CNP in vitro. In transgenic rats, NPR-B⌬KC expression selectively reduced NPR-B but not NPR-A signaling. NPR-B⌬KC transgenic rats display progressive, blood pressure-independent cardiac hypertrophy and elevated heart rate. The hypertrophic phenotype is further enhanced in chronic volume overload-induced congestive heart failure. Thus, this study provides evidence linking NPR-B signaling to the control of cardiac growth.

Fig. 2. Transgenic construct and NPR-B⌬KC mRNA expression. (A) Structure of NPR-B⌬KC transgenic construct. 1 and 2 indicate primers used to generate the probe for NPR-B兾NPR-B⌬KC ribonuclease protection assay. Primer 2 matched to NPR-B intracellular domain not present in NPR-B⌬KC. CMV, cytomegalovirus promoter for ubiquitous expression of the transgene; ␤-globin, rabbit ␤-globin intron for enhanced expression of the transgene; TM, transmembrane domain. (B) Expression of NPR-B and NPR-B⌬KC mRNA in the aorta and four heart chambers of transgenic rats shown by ribonuclease protection assay.

Fig. 1. NPR-B⌬KC acts as NPR-B-specific dominant-negative mutant in vitro. (A) Principle of NPR-B activation and inhibition. (Left) Native receptor. (Right) Dominant-negative mutant. (B) CNP-dependent cGMP response in COS cells cotransfected with NPR-B and various amounts of NPR-B⌬KC. *, P ⬍ 0.05; **, P ⬍ 0.01 vs. control by ANOVA; #, P ⬍ 0.05 vs. NPR-B native by ANOVA (n ⫽ 6 per group). (C and D) Measurement of cGMP response to ANP (C) or CNP (D) of H9c2 cells. **, P ⬍ 0.01 vs. control (n ⫽ 6 per group). (E and F) L-(4,53H)leucine incorporation of NPR-B⌬KC-transfected H9c2 cells and controls after stimulation with AVP (E) or IGF-1 (F) and treatment with ANP, CNP, or 8-Bromo-cGMP. **, P ⬍ 0.01; ***, P ⬍ 0.001 vs. single treatment with hypertrophic stimulus; ##, P ⬍ 0.01 vs. control AVP plus CNP; ###, P ⬍ 0.001 vs. control IGF-1 plus CNP (n ⫽ 6 per group).

Results NPR-B⌬KC Acts as a Dominant-Negative Molecule in a NPR-B-Specific Manner. COS-7 cells, which do not express NPR-A or NPR-B, were

cotransfected with NPR-B and the NPR-B deletion mutant NPRB⌬KC (Fig. 1A). Intracellular cGMP accumulation upon CNP stimulation was abolished with increasing amounts of NPR-B⌬KC, demonstrating a dominant-negative effect of the receptor mutant (Fig. 1B). NPR-B⌬KC was stably overexpressed in H9c2 cells, which express NPR-A and NPR-B (20) and respond to ANP or CNP stimulation with accumulation of intracellular cGMP in a concentration-dependent fashion. The cGMP response to ANP was unaffected by NPR-B⌬KC expression; however, the CNP-dependent cGMP response was significantly blunted (Fig. 1 C and D), demonstrating NPR-B specificity of NPR-B⌬KC in the presence of endogenous NPR-B. NPR-B Mediates Antihypertrophic Effects of CNP in H9c2 Cells. H9c2

cells, stably transfected with NPR-B⌬KC, were used to study the role of NPR-B signaling on cellular hypertrophy. IGF-1 (10⫺7 M) and Arg-vasopressin (AVP; 10⫺7 M) induced protein synthesis in H9c2 cells, accompanied by an increase in cell size (data not shown). ANP (10⫺7 M) and the membrane-permeable cGMP 4736 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0510019103

analogue 8-Bromo-cGMP (10⫺4 M) were equally potent in blocking IGF-1- and AVP-induced hypertrophy in both NPR-B⌬KCtransfected cells and controls (Fig. 1 E and F). In contrast, CNP was significantly less potent in reducing IGF-1- and AVP-induced hypertrophy in NPR-B⌬KC-expressing cells, suggesting antihypertrophic effects of NPR-B signaling in vitro. Generation and Characterization of NPR-B⌬KC Transgenic Rats.

Transgenic rats carrying the dominant-negative mutant NPRB⌬KC were generated to study the function of NPR-B in vivo (Fig. 2A and Fig. 7A, which is published as supporting information on the PNAS web site). NPR-B⌬KC transcripts were detected in all tested tissues of transgenic rats by RT-PCR (Fig. 7B). Because this study focused on the role of NPR-B in the cardiovascular system, expression of NPR-B and NPR-B⌬KC mRNA was quantified and shown to be equal in aorta, atria, and ventricles of transgenic and wild-type animals (Fig. 2B). To test whether NPR-B⌬KC was able to block the endogenous NPR-B in vivo, we measured the cGMP response to ANP and CNP in primary cells and tissues derived from NPR-B⌬KC transgenic rats. Expression of the transgene in cells used for the experiment was confirmed by RT-PCR (Fig. 3A and Fig. 8A, which is published as supporting information on the PNAS web site). Neonatal CMCs isolated from wild-type controls responded to ANP and CNP stimulation with a dose-dependent cGMP increase. The response to ANP stimulation of CMCs from NPR-B⌬KC transgenic rats was comparable to wild-type controls, whereas the cGMP response to CNP was attenuated (Fig. 3 B and C). Glomeruli isolated from transgenic rats exhibit reduced ligand-independent cGMP production at baseline. However, glomeruli from both transgenic rats and wild-type controls showed a comparable cGMP response upon stimulation with ANP, whereas the cGMP response to CNP was blunted in glomeruli from transgenic rats (Fig. 8). Aiming to further prove selective inhibition of NPR-B⌬KC on NPR-B signaling in an in vivo environment, ANP and CNP were infused i.v., and plasma cGMP concentrations were measured. Application of ANP (0.5 ␮g兾kg per min) resulted in comparable cGMP plasma values in NPR-B⌬KC transgenic rats and wild-type Langenickel et al.

Fig. 3. Reduced CNP-dependent cGMP generation in NPR-B⌬KC transgenic rats. (A) Expression of NPR-B and NPR-B⌬KC shown by RT-PCR in CMCs. (B and C) ANP-dependent (B) and CNP-dependent (C) cGMP production of CMCs from transgenic and wild-type rats. *, P ⬍ 0.05; **, P ⬍ 0.01 vs. wild-type control; #, P ⬍ 0.05 vs. wild-type control by ANOVA (n ⫽ 5– 6 per group). TG, transgenic rats; WT, wild-type control.

controls (wild type, 9.48 ⫾ 0.4 fmol兾ml; NPR-B⌬KC, 8.8 ⫾ 0.4 fmol兾ml). However, plasma concentrations of cGMP after CNP infusion (1.0 ␮g兾ml per min) were lower in NPR-B⌬KC transgenic rats (wild type, 9.2 ⫾ 0.9 fmol兾ml; NPR-B⌬KC, 6.3 ⫾ 0.8 fmol兾ml; P ⬍ 0.05), suggesting that NPR-B⌬KC acts as a dominant-negative mutant specifically interfering with NPR-B signaling in the presented transgenic model. Blood Pressure Regulation and Increased Heart Rate in NPR-B⌬KC Transgenic Rats. Telemetric blood pressure measurements showed

no significant differences in systolic, diastolic, and mean arterial pressure between NPR-B⌬KC transgenic rats and wild-type controls (Table 1). However, the heart rate of NPR-B⌬KC transgenic rats was found to be modestly elevated. Fourier transformation of systolic blood pressure, pulse interval time series, and cross spectra obtained by telemetry revealed increased sympathetic nerve activity in transgenic rats compared with wild-type controls, as shown by increased low frequency as measure of sympathetic heart rate control, increased low-frequency兾high-frequency ratio as a measure of sympathovagal balance, and increased power spectral density of systolic blood pressure calculated in the low-frequency band (Table 1). Normal Renal Function in NPR-B⌬KC Transgenic Rats. Assessment of renal function in metabolic cages revealed no influence of NPRB⌬KC on drinking behavior, natriuresis, diuresis, fractional sodium excretion, and creatinine clearance at baseline or after addition of 1% NaCl in drinking water or 1% NaCl in water plus 4% NaCl in the food (Fig. 9 A–D, which is published as supporting information on the PNAS web site). When the acute renal effect of ANP or CNP infusion was analyzed, both transgenic and wild-type control ani-

Fig. 4. Morphological assessment of cardiac hypertrophy in 6-month-old animals. (A) Hearts and hematoxylin and eosin-stained cardiac sections of wild-type (WT) and transgenic (TG) rats. Measurement of CMC diameter (B) and cross-sectional area (C). **, P ⬍ 0.01 vs. wild-type control (n ⫽ 5 hearts). Two hundred images per genotype were used for statistical analysis.

mals showed comparable dose-dependent diuretic responses to ANP, whereas diuresis did not significantly change after CNP infusion in either group (Fig. 9 E and F). Thus, in contrast to NPR-A, NPR-B signaling does not significantly contribute to regulation of renal function. Reduced Bone Growth in NPR-B⌬KC Transgenic Rats. As expected and

shown for the NPR-B-null mice, x-ray-based measurements revealed a reduced overall length (naso-anal length: wild type, 163.0 ⫾ 1.5 mm; NPR-B⌬KC, 150.0 ⫾ 2.0 mm; P ⬍ 0.01) and a reduction of the length of several bones (tibia: wild type, 27.5 ⫾ 0.2 mm; NPR-B⌬KC, 25.3 ⫾ 0.3 mm; P ⬍ 0.001; see Fig. 10, which is published as supporting information on the PNAS web site). No other obvious skeletal malformations were detected. Cardiac Hypertrophy in NPR-B⌬KC Transgenic Rats. Morphological and histological analysis of hearts from 6-month-old NPR-B⌬KC transgenic and wild-type animals revealed a concentric left ventricular hypertrophy along with increased CMC diameter and cross-sectional area in the transgenic rats (Fig. 4). In addition, left ventricular expression of ANP and BNP mRNA (Fig. 11, which is published as supporting information on the PNAS web site), along with ANP and BNP plasma concentrations (ANP: wild type, 71.9 ⫾ 11.6 fmol兾ml; NPR-B⌬KC, 125.7 ⫾ 21.8 fmol兾ml; P ⬍ 0.05; BNP: wild type, 15.9 ⫾ 0.9 fmol兾ml; NPR-B⌬KC, 19.9 ⫾ 0.7 fmol兾ml; P ⬍ 0.01), were found to be elevated in transgenic rats. The degree of interstitial and perivascular fibrosis, as assessed by

Rats Wild-type control (n ⫽ 10) NPR-B⌬KC transgenic (n ⫽ 10)

Mean arterial pressure, mmHg

Systolic blood pressure, mmHg

Diastolic blood pressure, mmHg

Heart rate, bpm

LF-RR, msec2

HF-RR, msec2

LF兾HF-RR

SBP-LF, mmHg2

105.0 ⫾ 1.2 105.9 ⫾ 0.7

122.5 ⫾ 1.6 125.4 ⫾ 1.6

89.9 ⫾ 1.0 88.0 ⫾ 0.8

322 ⫾ 4.1 339 ⫾ 2.4**

0.64 ⫾ 0.15 2.17 ⫾ 0.59*

1.55 ⫾ 0.22 2.54 ⫾ 0.31*

0.40 ⫾ 0.06 0.79 ⫾ 0.14*

2.20 ⫾ 0.19 3.47 ⫾ 0.40*

LF-RR, low-frequency power; HF-RR, high-frequency power; LF兾HF, ratio for heart rate variability; SBP-LF, low-frequency power of systolic blood pressure variability. *, P ⬍ 0.05, **, P ⬍ 0.01 vs. wild-type control. Langenickel et al.

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Table 1. Hemodynamic characterization in conscious rats under baseline conditions

Fig. 5. Echocardiography of 3- and 12-month-old animals. (A and B) Measurements of interventricular septum (IVS dia), posterior wall thickness (PWT dia), and calculated ratio of posterior wall thickness over left ventricular end-diastolic diameter (PWT dia兾LVEDD) of 3-month-old rats (A; n ⫽ 8 per group) and 12-month-old rats (B; n ⫽ 10 per group). *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001 vs. wild-type control. TG, transgenic rats; WT, wild-type control. (C and D) Left ventricular end-diastolic diameter (LVEDD), end-systolic diameter (LVESD), and ejection fraction (EF%) of 3-month-old rats (C) and 12month-old rats (D). ***, P ⬍ 0.001 vs. wild-type control.

Masson’s trichrome staining, was not different in transgenic rats from controls (Fig. 12, which is published as supporting information on the PNAS web site). Also, left ventricular mRNA levels of collagen 1 and 3 were not different between the groups (collagen 1: wild type, 0.77 ⫾ 0.02 arbitrary units; NPR-B⌬KC, 0.83 ⫾ 0.01 arbitrary units; collagen 3: wild type, 0.74 ⫾ 0.01 arbitrary units; NPR-B⌬KC, 0.77 ⫾ 0.01 arbitrary units; Fig. 11). Echocardiographic assessment of cardiac function in NPRB⌬KC transgenic rats and wild-type controls demonstrated early concentric left ventricular hypertrophy but no dilatation at 3 months of age, with increased interventricular septum and posterior wall thickness but unchanged end-diastolic and end-systolic diameters of the left ventricle (Fig. 5 A and C). Left ventricular contractile function was not altered by transgenic expression of NPR-B⌬KC, as demonstrated by unchanged EF (Fig. 5C). Cardiac hypertrophy was further enhanced in 12-month-old NPR-B⌬KC transgenic rats, as demonstrated by increased intraventricular septum and posterior wall thickness and further increased ratio of these parameters, which accounts for hypertrophy (Fig. 5B). Left ventricular end-diastolic and end-systolic diameters were decreased, whereas left ventricular contractility was increased in transgenic rats compared with wild-type controls (Fig. 5D). The relative heart weight of NPR-B⌬KC transgenic rats at 12 months of age was significantly increased (wild type, 3.02 ⫾ 0.05 mg兾g of body weight; NPR-B⌬KC, 3.35 ⫾ 0.13 mg兾g of body weight; P ⬍ 0.01). Augmented Cardiac Hypertrophy and Maintained Left Ventricular Function in NPR-B⌬KC Transgenic Rats After Chronic Volume Overload.

Chronic volume overload led to a significantly pronounced increase in cardiac hypertrophy in NPR-B⌬KC transgenic rats compared with wild-type controls measured echocardiographically 6 weeks after induction of an aortocaval shunt (Fig. 6A). In comparison to wild-type controls, left ventricular dilatation was less pronounced and left ventricular ejection fraction was less attenuated in NPRB⌬KC transgenic rats (Fig. 6B). Discussion In the present article we studied the cardiovascular function of the NPR-B receptor in vitro and in vivo. Employing a genetic 4738 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0510019103

Fig. 6. Echocardiography of 14-week-old rats with chronic volume overload 6 weeks after surgery. (A) Measurements of interventricular septum (IVS dia), posterior wall thickness (PWT dia), and calculated ratio of posterior wall thickness over left ventricular end-diastolic diameter (PWT dia兾LVEDD). **, P ⬍ 0.01; ***, P ⬍ 0.001 vs. sham-operated animals; ##, P ⬍ 0.01; ###, P ⬍ 0.001 vs. wild-type shunt animals; $, P ⬍ 0.05 vs. sham-operated wild-type animals (n ⫽ 7–10 per group). TG, transgenic rats; WT, wild-type control. (B) Left ventricular end-diastolic diameter (LVEDD), end-systolic diameter (LVESD), and ejection fraction (EF%). ***, P ⬍ 0.001 vs. sham-operated animals; ###, P ⬍ 0.001 vs. wild-type shunt animals; $, P ⬍ 0.05; $$, P ⬍ 0.01 vs. sham-operated wild-type animals (n ⫽ 7–10 per group).

model overexpressing a dominant-negative NPR-B mutant in transgenic rats, we demonstrate for the first time that NPR-B signaling is involved in regulation of cardiac hypertrophy. The dominant-negative properties of NPR-B⌬KC were confirmed by a gene dose-dependent reduction of NPR-B signaling by NPR-B⌬KC in COS-7 and H9c2 cells as well as in transgenic rats ubiquitously expressing NPR-B⌬KC, as shown by attenuated cGMP response after CNP infusion in intact animals or CNP stimulation of adult CMCs and glomeruli ex vivo. These findings mirror data published by Chinkers and Wilson (21) demonstrating a lack of interaction between the extracellular domains of NPR-A and NPR-B. Furthermore, a dominant-negative mutant of NPR-A as well as an alternative splice variant of NPR-B acting in a dominant-negative manner have previously been used to study ANP and CNP physiology, providing evidence for the use of dominant-negative mutants in cell culture and transgenic animal models as a powerful method to study the specific function of NPRs (19, 22–24). Several other genetically altered models of the NP system provided evidence for antihypertrophic actions of NP (11, 12, 14, 25). Both ANP and CNP were capable of reducing the hypertrophic response to a broad range of hormonal or mechanical stimuli in several cell types, including CMCs, cardiac fibroblasts, and vascular smooth muscle cells (8, 17, 26). This effect was mediated by their second messenger cGMP and could be blocked by an unspecific NPR antagonist. However, because of a paucity of specific pharmacological inhibitors or a suitable genetic model, none of these previous studies could clearly define the role of NPR-B in cardiac hypertrophy. In accordance with the blunted CNP response in NPR-B⌬KCtransfected H9c2 cells, we found a reduced potency of CNP, but not Langenickel et al.

Langenickel et al.

study of cardiovascular functions of NPR-B. Employing this model, we showed for the first time that NPR-B mediates antihypertrophic effects in CMCs under physiological as well as pathophysiological conditions. Further studies need to address the signaling pathway involved in mediating these effects of CNP兾NPR-B. Methods Cell Culture and in Vitro Studies. H9c2 and COS-7 cells were

obtained from American Type Culture Collection and were cultivated as described in ref. 20. Rat NPR-B cDNA was a gift from Lincoln Potter (University of Minnesota, Minneapolis). NPRB⌬KC cDNA was cloned into pTarget (Promega) or pcDNA3.1(-) -myc兾His (Invitrogen). For titration experiments, COS-7 cells were cotransfected with different ratios of pcDNA3.1兾NPR-B and pcDNA3.1兾NPR-B⌬KC. H9c2 cells were stably transfected with pcDNA3.1兾NPR-B⌬KC. Transfected cells were serum-starved for 24 h followed by stimulation with 10⫺7 M AVP or 10⫺7 M IGF-1 with and without 10⫺7 M ANP, 10⫺7 M CNP, or 10⫺4 M 8-BromocGMP (all from Calbiochem), in the presence of 1 ␮Ci兾ml (1 Ci ⫽ 37 GBq) L-(4,5-3H)leucine (Amersham Pharmacia). After 24 h, radioactive labeling was measured by liquid scintillation counting. Generation of Transgenic Rats. pTarget-NPR-B⌬KC was linearized and microinjected into the male pronucleus of rat zygotes as described (37). Founder animals were identified by Southern blotting by using 32P-labeled NPR-B⌬KC as a probe and bred to homozygosity. Further genotyping was conducted by PCR (for primers see Table 2, which is published as supporting information on the PNAS web site). Analysis of mRNA Expression. After rats were killed, organs were

dissected, frozen in liquid nitrogen, and stored at ⫺80°C. Total RNA was extracted by using TRIzol reagent (Invitrogen). Antisense probes for NPR-B⌬KC, ANP, BNP, collagen 1 and 3, and GAPDH (for primers see Table 2) were 32P-UTP-labeled by in vitro transcription (Riboprobe combination system Sp6兾T7; Promega). RNA expression was measured by RNase protection assay (RPA II kit, Ambion). Protected fragments were separated on a polyacrylamide gel and detected with a Fuji phosphoimager (Fujix BAS 2000, Fuji).

Isolation and Stimulation of Neonatal CMCs and Glomeruli. Neonatal rat CMCs were obtained from 2- to 3-day-old rats and cultured as described (38). Glomeruli were isolated by differential sieving as published (39). CMC and glomeruli were stimulated at 37°C for 20 min with different concentrations of ANP or CNP in the presence of 0.5 mM 3-isobutyl-1-methylxanthine (Sigma). Samples were kept at ⫺20°C until cGMP measurement. Analysis of ANP, BNP, and cGMP. Plasma samples for ANP and BNP

were purified on C18 Sep-Pack columns (Waters), and peptide concentrations were determined by using RIA kits (Bachem). For cGMP analysis, plasma and glomeruli samples were purified on alumina matrix (Sigma). Supernatant from CMC was directly used for the assay. cGMP values were normalized by protein concentration. For experiments with H9c2 and COS-7 cells, intracellular cGMP was analyzed. Determination of cGMP was performed by RIA as described in ref. 40. Assessment of Renal Function. Renal excretory function in conscious

rats was assessed by using metabolic cages. Diuresis and water intake were monitored under baseline conditions, with 1% NaCl loading in drinking water or 1% NaCl in water plus 4% NaCl in food for 24 h, respectively. The concentrations of sodium and creatinine in urine and plasma were measured by Labor Diagnostik (Leipzig, Germany), and fractional sodium excretion, and creatinine clearance were calculated. The renal effect of acute ANP and CNP infusion was analyzed under isoflurane anesthesia. Briefly, the PNAS 兩 March 21, 2006 兩 vol. 103 兩 no. 12 兩 4739

PHYSIOLOGY

ANP, to block AVP- or IGF-1-induced hypertrophy in H9c2 cells. These data show that the effects of CNP were mediated by NPR-B and cannot be attributed to crossactivation of NPR-A by CNP because of supraphysiological concentrations used in cell culture. Histological assessment and echocardiography revealed cardiac hypertrophy in NPR-B⌬KC transgenic rats, which aggravated with age accompanied by increased cardiac ANP and BNP mRNA expression and increased ANP and BNP plasma concentration. However, there was no evidence for increased interstitial or perivascular fibrosis. Furthermore, chronic volume overload by an infrarenal aortocaval shunt in 8-week-old rats resulted in exaggerated cardiac hypertrophy in NPR-B⌬KC transgenic rats 6 weeks after surgery. These findings suggest that NPR-B is implicated in the regulation of CMC growth but not in cardiac fibrosis. The increased left ventricular contractility observed in 12-monthold transgenic animals and the less affected left ventricular function in the transgenic rats after aortocaval shunt could result from cardiac hypertrophy (27) or from inhibition of NPR-B signaling. Pierkes et al. (28) demonstrated a negative inotropic effect after initial positive inotropic actions of CNP in isolated mouse working hearts, which could contribute to the observed increase in ejection fraction as a consequence of chronic inhibition of CNP signaling in NPR-B⌬KC transgenic rats. Given the possibility of involvement of NPR-B in blood pressure regulation, we aimed to elucidate this function using telemetry in conscious animals. Blood pressure was not different, but the heart rate was moderately elevated in transgenic rats compared with controls. Fourier transformation and power spectral analysis of the data obtained from these telemetric measurements suggest an increased sympathetic nerve activity in transgenic rats. A possible mechanism could be a reduced central nervous CNP action to facilitate vagal heart rate baroreflexes by inhibition of NPR-B (29). Because the increase in heart rate observed in our model is rather small it is unlikely to cause cardiac hypertrophy over a period as short as 3 months, when hypertrophy was first detected. Our findings do not confirm previous studies that predicted a role of CNP in blood pressure regulation by i.v. administration of supraphysiological CNP concentrations (30) or by exposing isolated vessels to CNP (31, 32), but they are in accordance with the phenotype of NPR-B-null mice, where no blood pressure differences were reported (19). We conclude that normal levels of CNP are most likely not involved in basic blood pressure regulation in vivo and that the observed cardiac hypertrophy is blood pressure-independent. We found that down-regulation of NPR-B signaling did not affect renal function parameters at baseline or after dietary salt load. Also, in contrast to ANP, CNP infusion did not induce diuresis in wild-type animals. This finding is in accordance with previously published data showing that CNP does not influence renal function in humans and dogs after i.v. administration at low dosages (33, 34). It has been demonstrated that systemic infusion or bolus administration of supraphysiological CNP concentrations in dogs increased aldosterone release and distal nephron sodium reabsorption, suggesting that CNP regulates tubular reabsorption and not glomerular filtration rate (35). Despite conflicting data on acute renal effects of CNP, fluid homeostasis in chronic down-regulation on NPR-B signaling appears to be balanced. Thus, we suggest that NPR-B is not a major regulator of renal function in vivo. Longitudinal growth of the long bones is a result of endochondral ossification in the cartilaginous growth plate. We report in the present study a growth retardation of long bones in NPR-B⌬KC transgenic rats, supporting published data on the role of NPR-B in endochondral ossification (18, 19, 36). In summary, the present study demonstrates that a mutant of NPR-B lacking a substantial part of the cytoplasmatic domain acts as selective inhibitor of the native NPR-B in vitro and in vivo. In contrast to CNP- and NPR-B-deficient mice, which are severely ill, NPR-B⌬KC transgenic rats have been proven very valuable for the

jugular vein was cannulated. After 20 min of equilibration, ANP (0.5 ␮g兾kg per min) or CNP (1.0 ␮g兾kg per min) were infused over 20 min. Urine was collected over three time periods of 20 min each for calculation of diuresis, and plasma samples were stored at ⫺80°C for analysis of cGMP excretion. After washout, the experiment was repeated with a higher dosage of ANP (1.0 ␮g兾kg per min) or CNP (2.0 ␮g兾kg per min). Transthoracic Echocardiography, Hemodynamic Evaluation, and Spectral Analysis of Autonomic Nervous System. Transthoracic

two-dimensional guided M-mode echocardiography was performed in 3- and 12-month-old rats under anesthesia with isoflurane, by using an Acuson Sequoia C256 echocardiograph with a 14-MHz probe (Siemens). For radiotelemetric blood pressure measurements in conscious animals, 10 rats per group were anesthetized with ketamine兾 xylazine. Pressure transducers were implanted in the abdominal cavity, and the transducer-connected catheter was anchored in the lumen of the abdominal aorta. After recovery for 15 days, data were recorded under baseline conditions. Autonomic nerve activity was assessed by monitoring spontaneous changes in blood pressure and heart rate as described earlier (41).

Aortocaval Shunt. The infrarenal aortocaval shunt was used as a

model of volume overload-induced heart failure in 8-week-old rats as described (40). Sham-operated rats of each genotype were used as controls. Six weeks after surgery, sham- and shunt-operated rats were used for echocardiography and molecular biology studies. All animal studies were carried out in accordance with the local authorities and conforming to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 85-23, revised 1996). Skeletal Phenotyping. Thirteen-month-old rats were anesthetized

with ketamine兾xylazine and underwent soft x-ray examination (Polydoros LX 50 generator; Siemens). Statistical Analysis. Differences between two groups were evaluated by using unpaired Student’s test, and differences between more than two groups were evaluated by using ANOVA followed by Fisher’s probable least-squares difference test using STATVIEW software. ANOVA for repeated measurements was applied to evaluate cellular cGMP responses and diuresis after ANP and CNP infusion. The significance level was set at P ⬍ 0.05. All data are expressed as mean ⫾ SEM.

animals were placed in 10% formaldehyde and embedded in paraffin. Longitudinal and transversal sections (5 ␮m) were stained with hematoxylin and eosin or Masson’s trichrome. CMC diameter and cross-sectional area were assessed at ⫻40 magnification.

We gratefully acknowledge the excellent technical assistance of Astrid Schiche, Jutta Meisel, Jeannette Mothes, Rita Gu ¨nzel, Rosemarie Barnow, and Reika Langanki. This project was supported by grants from ¨ck Center for Molecular Medicine Berlin-Buch (to the Max Delbru T.H.L., I.P.-L., M.B., J.M., and J.B.) and by a fellowship from the Charite´ Medical School (to J.B.).

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Histological Analysis. Hearts from 6-month-old transgenic or control

4740 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0510019103

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