Cardiovascular Research

Cardiovascular Research 2003 57(2):515-522; doi:10.1016/S0008-6363(02)00667-3
© 2003 by European Society of Cardiology

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Copyright © 2003, European Society of Cardiology

Antihypertrophic actions of the natriuretic peptides in adult rat cardiomyocytes: importance of cyclic GMP

Anke C Rosenkranz, Robyn L Woods, Gregory J Dusting and Rebecca H Ritchie^*

Howard Florey Institute, c/- The University of Melbourne, Parkville VIC 3010, Australia

^* Corresponding author. Tel.: +61-383-445-654; fax: +61-393-481-707 r.ritchie{at}hfi.unimelb.edu.au

Received 5 June 2002; accepted 11 September 2002

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Atrial natriuretic peptide (ANP) prevents hypertrophy of neonatal cardiomyocytes. However, whether this effect is retained in the adult phenotype or if other members of the natriuretic peptide family exhibit similar antihypertrophic properties, has not been elucidated. Objective: Our objective was to examine whether the natriuretic peptides protect against adult cardiomyocyte hypertrophy in vitro. Methods: Adult rat cardiomyocytes were incubated with angiotensin II (Ang II)±ANP, B-type (BNP) or C-type (CNP) natriuretic peptides for determination of [³H]phenylalanine incorporation, c-fos mRNA expression and cyclic GMP. The effects of 8-bromo-cyclic GMP (cyclic GMP analogue), HS-142-1 (particulate guanylyl cyclase inhibitor) and KT5823 (cyclic GMP-dependent protein kinase inhibitor) were also investigated. Results: Ang II-stimulated increases in markers of hypertrophy, [³H]phenylalanine incorporation (to 136±3% of control, n=9) and c-fos mRNA expression (4.3±1.4-fold, n=5), were completely prevented by each of ANP, BNP or CNP. This protective action was accompanied by increased cardiomyocyte cyclic GMP. Inhibitory actions on [³H]phenylalanine incorporation were mimicked by 8-bromo-cyclic GMP, and were abolished by HS-142-1. KT5823 blocked the response to BNP and CNP, but not to ANP. Conclusion: ANP prevents hypertrophy of adult rat cardiomyocytes. This protective action is shared by BNP and CNP and involves activation of particulate guanylyl cyclase receptors. Antihypertrophic effects of BNP and CNP are mediated through cyclic GMP-dependent protein kinase, but ANP can activate additional pathways independent of cyclic GMP to prevent adult cardiomyocte hypertrophy. These novel findings are of interest particularly since BNP appears to exert antifibrotic rather than antihypertrophic actions in vivo [12], while CNP is thought to act at least in part via the endothelium [8].

KEYWORDS Angiotensin; Hypertrophy; Natriuretic peptide; Second messengers

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Cardiac hypertrophy develops to preserve contractile function when cardiac workload is chronically increased [1], but in the longer term is associated with increased cardiovascular risk [2]. At a cellular level, cardiomyocyte hypertrophy is characterised by increased protein synthesis and cell size, and induction of immediate early (e.g. c-fos, c-jun) and foetal genes (e.g. atrial natriuretic peptide, β-myosin heavy chain) [3]. Atrial natriuretic peptide has been shown to inhibit hypertrophy of neonatal cardiomyocytes through a cyclic GMP-dependent process involving particulate guanylyl cyclase receptors [4,5]. A similar action has not been investigated in the adult cardiomyocyte, and it is unknown if the antihypertrophic effect of ANP is shared by the other members of the natriuretic peptide family, the B-type (BNP) and C-type (CNP) natriuretic peptides.

ANP and BNP are circulating cardiac hormones [6,7], while CNP is produced primarily by vascular endothelium and is thought to act locally as a paracrine regulator of tone [8]. Expression of the three identified natriuretic peptide receptors (NPR-A, NPR-B, NPR-C) has been confirmed in human and rat cardiac tissue [9]. Both NPR-A, preferentially activated by ANP and BNP, and NPR-B, selective for CNP, are coupled to a cytoplasmic C-terminal guanylyl cyclase catalytic domain and signal via formation of cyclic GMP. The clearance receptor NPR-C, which binds all three natriuretic peptides, is not coupled to particulate guanylyl cyclase. Deficiency of either ANP or NPR-A is associated with cardiac hypertrophy [10–12] but in contrast BNP –/– mice develop massive fibrotic lesions with no evidence of cardiac enlargement [13,14]. Thus while both BNP and CNP share the antiproliferative action of ANP in vascular smooth muscle cells [15] it is unclear whether the same is true for the prevention of cardiomyocyte hypertrophy, particularly since CNP activates a different receptor, still coupled to particulate guanylyl cyclase.

The objective of the present study was therefore to examine the antihypertrophic actions of all three natriuretic peptides, ANP, BNP and CNP, in adult rat cardiomyocytes. Mature cells are less likely to de-differentiate and do not exhibit the marked upregulation of ANP, contractile proteins or other changes seen in the neonatal phenotype [3,16], and hypertrophic signalling is thus more likely to reflect the in vivo situation.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
2.1 Materials
Ang II, 8-bromo-cyclic GMP and 3-isobutyl-methylxanthine (IBMX), were purchased from Sigma (St. Louis, United States). KT5823 was obtained from Calbiochem-Novabiochem (La Jolla, United States). HS-142-1 was a generous gift from Dr Satoshi Nakanishi, Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co. Ltd., Shizuoka-ken, Japan. The natriuretic peptides (rat ANP-28, rat BNP-32, and human, porcine, rat CNP-22) were from Bachem Feinchemikalien (Bubendorf, Switzerland). All reagents for cell culture were of tissue culture grade. All other materials were purchased from Sigma except where indicated, and were of analytical grade or higher. Real Time PCR (fluorogenic probes, TaqMan^® Universal PCR master mix and TaqMan^® RT reagents) reagents were all purchased from Applied Biosystems (Scoresby, Australia).

2.2 Isolation of rat cardiomyocytes
Male Sprague–Dawley rats (180–280 g) were anaesthetised intraperitoneally with ketamine hydrochloride (100 mg/kg) and xylazine (12 mg/kg) and hearts rapidly removed. Cardiomyocytes were freshly isolated as previously described [17] and resuspended in serum-free bicarbonate-buffered medium 199 (M199, with Earle's Salts and 25 mmol/l HEPES, Trace Scientific, Australia), supplemented with 0.2% albumin (bovine fraction V), 2 mmol/l L-carnitine, 5 mmol/l creatine, 5 mmol/l taurine, 25 µg/ml gentamicin (Life Technologies, New York, United States), 100 U/ml penicillin and 100 µg/ml streptomycin (CSL Biosciences, Australia). Cardiomyocytes were either suspended in six-well plates (Falcon/Becton Dickinson, Lincoln Park, United States) for measurement of c-fos mRNA or intracellular cyclic GMP content (5x10⁵ cells/35 mm well), or plated onto laminin (10 µg/ml; Collaborative Biomedical Products, Bedford, United States)-coated six-well plates for [³H]phenylalanine incorporation (6x10⁴ cells/well). Cells were incubated at 37 °C (5% CO₂ in air) until required (2–24 h). This technique yields <7% nonmyocyte contamination, as previously described [17]. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publications No. 85-23, revised 1996) and was approved by the Animal Experimentation Ethics Committee of the Howard Florey Institute.

2.3 [³H]Phenylalanine incorporation in cardiomyocytes
[³H]Phenylalanine incorporation by cardiomyocytes was determined as an in vitro marker of hypertrophy. Following equilibration, incubation medium was replaced with serum-free M199 containing 2 µCi/ml of L-[2,3,4,5-³H]-phenylalanine (NEN Lifesciences, Boston, MA, USA)±Ang II (1 µmol/l)±study drugs, and cardiomyocytes were incubated for 2 h at 37 °C. Study drugs included the cyclic GMP analogue 8-bromo-cyclic GMP (1 mmol/l), the NO donor SNP (30 µmol/l), or the natriuretic peptides ANP, BNP or CNP (each 1 µmol/l). A range of natriuretic peptide concentrations (10 nmol/l–10 µmol/l) was investigated in preliminary concentration-effect studies (n=3) to determine the minimal effective concentration. The influence of the dual NPR-A/NPR-B receptor antagonist HS-142-1 (100 µg/ml) [5,18] was also examined, as was an inhibitor of cyclic GMP-dependent protein kinase, KT5823. The concentration of KT5823 used (250 nmol/l) was chosen to reflect the K_i for specific inhibition of cyclic GMP-dependent protein kinase (234 nmol/l) [19]. For KT5823 studies, all samples were supplemented with equivalent volumes of DMSO as vehicle control. Cardiomyocyte protein and DNA were trichloroacetic acid-precipitated prior to resuspension in 0.3 mol/l sodium hydroxide. [³H]Phenylalanine was determined by liquid scintillation counting. DNA content was determined fluorimetrically using PicoGreen^® reagent (Molecular Probes, Eugene, OR, USA). Results for [³H]phenylalanine incorporation were expressed relative to ng DNA/sample to correct for number of cells per sample [17].

2.4 Expression of c-fos mRNA by quantitative Real Time PCR
Real-Time Polymerase Chain Reaction (PCR) was used to measure mRNA expression of the immediate early gene c-fos as an additional marker of cardiomyocyte hypertrophy [20,21]. Cardiomyocytes were incubated for 45 min with serum-free M199 alone or with ANP, BNP or CNP (1 µmol/l). Ang II (1 µmol/l) was present during the final 15 min. Total RNA was extracted in 500 µl cold RNAWiz^TM (Ambion, TX, USA) following manufacturer's instructions. Total RNA was reverse-transcribed to a final cDNA concentration of 10 ng/µl using TaqMan^® reverse transcription (RT) reagents (Applied Biosystems). Reverse transcription was performed at 48 °C for 30 min followed by RT inactivation at 95 °C for 5 min (Perkin-Elmer GeneAmp 9600).

Real time PCR was used to determine c-fos mRNA expression using an ABI Prism^® 7700 sequence detection system (Applied Biosystems), which measures degradation of a dual-labelled fluorescent hybridisation probe in ‘real time’ [22,23] simultaneously with PCR amplification of product. Primers and fluorogenic probes for rat c-fos mRNA and the housekeeping gene (constitutively expressed in all tissues) 18S ribosomal RNA were constructed from rat-specific sequences published on GenBank (Table 1). Fluorogenic probes were labelled at the 5'-end with the reporter dye of FAM (c-fos) or VIC (18S) and at the 3'-end with the quencher molecule TAMRA. Both c-fos and 18S were amplified in the same tube to determine relative increases in c-fos transcripts relative to 18S. The final PCR reaction mix (total volume 25 µl) contained 5 ng cDNA template, 1 X TaqMan^® Universal PCR master mix (Applied Biosystems), as well as optimal concentrations of probes and forward and reverse primers (Table 1). Thermal cycler conditions were 2 min at 50 °C, 10 min at 95 °C and 40 cycles of 95 °C for 30 s and 60 °C for 1 min. Amplification of product, detected as increasing fluorescence, was plotted against PCR cycle number to determine the threshold cycle number (C_t), the point at which PCR product is detectable. To express c-fos mRNA expression relative to 18S mRNA, 18S C_t was subtracted from c-fos C_t (C_t). The fold increase in c-fos mRNA was then calculated by the C_t method [23]; fold induction of c-fos in each sample =2^–[C_t], where C_t is defined as C_t for that sample, minus the control group C_t. Fold induction of c-fos expression in the control sample is thus set as 1.0.

View this table: [in this window] [in a new window]   Table 1 Optimal sequences (5'3') and concentrations of Taqman primers and probes

 
2.5 Accumulation of cyclic GMP
Cardiomyocytes were incubated alone for 30 min in medium supplemented with IBMX (1 mmol/l, to prevent degradation of cyclic GMP)±Ang II (1 µmol/l)±ANP, BNP or CNP (all 1 µmol/l). Cardiomyocytes were rapidly washed with ice-cold phosphate-buffered saline (pH 7.4) prior to precipitation in 1.0 ml ice-cold 100% methanol. Well contents were scraped into glass vacutainer^® tubes (Becton Dickinson, USA) and dried. Pellets were frozen at –20 °C until resuspended in 300 µl sodium acetate buffer (pH 6.5) for measurement of cyclic GMP content using a commercial [¹²⁵I]-cyclic GMP radioimmunoassay (NEN Lifescience Products) [17]. Cyclic GMP content was determined as fmol cyclic GMP/100 µl sample.

2.6 Statistical analysis
Within each experiment, each treatment group was studied at least in triplicate and the average result taken. The ‘n’ value for each set of results represents the number of myocyte preparations studied. Data were normalised (%) to the measurement of the averaged control wells for each experiment and expressed as data is presented as mean±standard error of the least square mean for treatment (between experiment variation). Comparative statistical analyses were performed using 2-way analysis of variance (ANOVA); paired two-tailed t-tests were applied to compare the effect of the hypertrophic stimulus Ang II with paired controls, or where indicated. The Bonferroni correction for multiple contrasts was applied where appropriate. Values of P<0.05 were accepted as significant.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1 Natriuretic peptides protect cardiomyocytes from hypertrophy
The potential for antihypertrophic actions of all three natriuretic peptides was examined in isolated rat cardiomyocytes. A range of concentrations (10 nmol/l–10 µmol/l) was examined in initial studies (n=3) for each peptide to determine the optimal concentration for use in subsequent experiments. For all three natriuretic peptides, 1 µmol/l concentration provided near maximal inhibition of Ang II-induced increases in [³H]phenylalanine incorporation. The inhibitory effect of natriuretic peptides was inconsistent at lower concentrations (10 and 100 nmol/l) and was not enhanced by concentrations higher than 1 µmol/l (results not shown). For subsequent experiments, natriuretic peptides were examined at 1 µmol/l, and at this concentration the natriuretic peptides alone did not modulate [³H]phenylalanine incorporation (results not shown).

Each of the natriuretic peptides prevented Ang II-stimulated [³H]phenylalanine incorporation, from 136±3% of control (n=9 P<0.001 paired t-test) to 108±3% with ANP, 100±3% with BNP and 95±3% of control with CNP (all n=9, P<0.001 vs. Ang II, Fig. 1A). As an additional marker of hypertrophy, Ang II also increased c-fos mRNA expression by 4.3±0.7-fold (P<0.05, n=5, Fig. 1B) compared to control adult rat cardiomyocytes. The natriuretic peptides prevented this Ang II-stimulated increase in c-fos mRNA, to 1.02±0.7-fold with ANP, 1.10±0.7-fold with BNP and 0.86±0.7-fold with CNP (all n=5, Fig. 1B).

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 (A) Ang II (1 µmol/l, n=9)-stimulated increases in [3H]phenylalanine incorporation (% of control) were prevented by ANP (1 µmol/l), BNP (1 µmol/l) or CNP (1 µmol/l) in adult rat cardiomyocytes (all n=9). (B) ANP, BNP or CNP (1 µmol/l) also prevented Ang II-induced expression of c-fos mRNA in cardiomyocytes alone (all n=5). *P<0.05 vs. control (paired t-test), #P<0.05 vs. Ang II (2-way ANOVA), P<0.05 vs. control (2-way ANOVA).

 
3.2 Role of particulate guanylyl cyclase receptors
The influence of a guanylyl cyclase-coupled receptor antagonist, HS-142-1, on the antihypertrophic actions of the natriuretic peptides, was examined in cardiomyocytes. [³H]Phenylalanine incorporation was significantly stimulated by Ang II (127±7% of control, P<0.001, n=7). HS-142-1, which blocks NPR-A and NPR-B receptors, increased basal [³H]phenylalanine incorporation to a similar extent as Ang II (125±7% of control, n=7, P = NS vs. Ang II) but did not further increase Ang II-stimulated [³H]phenylalanine incorporation (134±7%, n=7, P = NS vs. Ang II, Fig. 2A). The antihypertrophic effects of ANP, BNP and CNP were significantly attenuated by HS-142-1 (Fig. 2B): [³H]phenylalanine incorporation was 125±4% (n=6, P<0.05 vs. Ang II+ANP), 133±10% (n=6, P<0.05 vs. Ang II+BNP), and 134±17% of control (n=6, P<0.05 vs. Ang II+CNP).

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 (A) An inhibitor of particulate guanylyl cyclase NPR-A and NPR-B receptors, HS-142-1 (100 µg/ml) stimulated basal [3H]phenylalanine incorporation in rat cardiomyocytes but did not further increase Ang II (1 µmol/l)-induced [3H]phenylalanine incorporation (all n=6). (B) HS-142-1 prevented the antihypertrophic actions of ANP, BNP and CNP (all 1 µmol/l, n=6) in rat cardiomyocytes. All data represented in Fig. 2B are paired with data in Fig. 2A and can be directly compared. *P<0.001 vs. control (paired t-test), P<0.05 vs. Ang II+natriuretic peptide (paired t-test).

 
3.3 Natriuretic peptides stimulate cardiomyocyte cyclic GMP
Ang II did not significantly alter cardiomyocyte cyclic GMP content (from 244±58 to 266±58 fmol/100 µl, n=6, Fig. 3). By contrast, the addition of ANP or BNP significantly stimulated cardiomyocyte cyclic GMP content, to 561±58 and 608±58 fmol/100 µl respectively (both P<0.005 vs. control, n=6, Fig. 3). CNP tended to increase cyclic GMP to 447±58 fmol/100 µl (n=6, Fig. 3); this trend was not significant (P=0.09).

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 ANP (n=6) and BNP (n=6) significantly stimulated intracellular cyclic GMP content (fmol/100 µl) in cardiomyocytes. CNP failed to significantly elevate cyclic GMP (P=0.09). *P<0.005 vs. control (2-way ANOVA).

 
3.4 Cyclic GMP as an antihypertrophic mediator in adult rat cardiomyocytes
The direct antihypertrophic action of cyclic GMP was also examined. Ang II-induced increases in [³H]phenylalanine incorporation (to 137±4% of control, P<0.001, n=6) were prevented by the stable cyclic GMP analogue 8-bromo-cyclic GMP (111±4% of control, n=6, P<0.05 vs. Ang II, Fig. 4A). 8-Bromo-cyclic GMP alone did not modulate [³H]phenylalanine incorporation (results not shown). The involvement of downstream cyclic GMP-dependent signalling in the antihypertrophic effect of natriuretic peptides was investigated using the selective cyclic GMP-dependent protein kinase inhibitor KT5823. Ang II significantly increased [³H]phenylalanine incorporation to 134±4% of control (P<0.001 vs. control, n=9, Fig. 4B). KT5823 did not influence basal [³H]phenylalanine incorporation (n=6) but abolished the antihypertrophic actions of BNP and CNP: [³H]phenylalanine incorporation was 136±10% (n=6, P<0.05 vs. Ang II+BNP) and 132±12% of control (n=6, P<0.05 vs. Ang II+CNP) in the presence of BNP+KT5823 and CNP+KT5823 respectively (Fig. 4B). In contrast, the ability of ANP to prevent Ang II-induced [³H]phenylalanine incorporation was not impaired by KT5823: [³H]phenylalanine incorporation was 108±14% of control (n=6, Fig. 4B). [³H]Phenylalanine incorporation in samples with Ang II alone or with Ang II+natriuretic peptide was not affected by the presence of DMSO.

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 (A) Ang II (n=6)-stimulated increases in [3H]phenylalanine incorporation (% of control, n=6) in cardiomyocytes were prevented by 8-bromo-cyclic GMP (1 mmol/l, n=6). (B) The cyclic GMP-dependent protein kinase inhibitor KT5823 (250 nmol/l) abolished the ability of BNP and CNP but not ANP to prevent Ang II stimulated increases in [3H]phenylalanine incorporation (all 1 µmol/l, n=6). KT5823 alone had no effect (n=6). *P<0.001 vs. control (paired-test), #P<0.005 vs. Ang II (paired t-test), P<0.05 vs. control (paired t-test).

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The present study demonstrates for the first time that ANP exerts an antihypertrophic action in adult rat cardiomyocytes, and furthermore that this protective effect is shared by the other natriuretic peptides, BNP and CNP. This finding is of particular interest given that neither BNP nor CNP had previously been examined in any model of cardiac hypertrophy. All three natriuretic peptides prevent Ang II-induced increases in both [³H]phenylalanine incorporation (Fig. 1A) and expression of c-fos mRNA (Fig. 1B). While the adult cardiomyocyte differs markedly from the neonatal phenotype, these findings are in agreement with the previous report in neonatal cardiomyocytes, where exogenous ANP at 0.1 µmol/l concentration attenuated noradrenaline (1 µmol/l)-induced [³H]leucine incorporation over 24 h, without influencing basal protein synthesis [4]. Conversely, while 0.1–1 µmol/l exogenous ANP failed to prevent phenylephrine (10 µmol/l)-induced protein synthesis by neonatal cardiomyocytes in another study, a clear antihypertrophic effect of endogenous ANP was demonstrated [5]. The observation that ANP exerts potent antihypertrophic actions in mature cardiomyocytes is therefore of particular interest.

In the present study, the finding that BNP and CNP elicit a similar antihypertrophic action in adult cardiomyoctes was also of great significance, given that CNP acts in part via the endothelium [8,24] and endogenous BNP does not appear to influence cardiac hypertrophy in vivo [13,14], despite activating the same particulate guanylyl cyclase receptor as ANP. It is likely that ANP is the more important antihypertrophic factor in vivo, with some contribution from BNP and CNP, although cardiomyocytes are able to respond to all three natriuretic peptides to counter hypertrophic stimuli. In support of this, disruption of the ANP/NPR-A pathway has deleterious consequences for cardiac hypertrophy [10,12,25], and we propose that increases in circulating natriuretic peptide prevent excessive cardiac enlargement during the earlier stages of hypertrophy. However, downregulation or uncoupling of natriuretic peptide receptors during the progression to decompensated heart failure [26,27] may ultimately limit the antihypertrophic action of the natriuretic peptides.

The antihypertrophic responses to all three natriuretic peptides were completely blocked by HS-142-1 (Fig. 2B), a dual NPR-A/NPR-B antagonist, indicating the essential role of particulate guanylyl cyclase-coupled receptors in the antihypertrophic response to natriuretic peptides. HS-142-1 also increased basal [³H]phenylalanine incorporation (Fig. 2A), as was previously reported in neonatal cardiomyocytes [5], and further supporting the role of the endogenous ANP/NPR-A system as a regulator of cardiac growth in vivo [11,12].

Activation of NPR-A and NPR-B receptors by natriuretic peptides stimulates cyclic GMP in a number of cell types [28], and in the present study, the natriuretic peptides, particularly ANP and BNP, were found to increase cardiomyocyte cyclic GMP content (Fig. 3). Importantly, the inhibitory action of natriuretic peptides on [³H]phenylalanine incorporation by cardiomyocytes is mimicked by 8-bromo cyclic GMP (Fig. 4A), in agreement with previous reports in neonatal cardiomyocytes [4]. Cyclic GMP may therefore be an important mediator of the antihypertrophic response to natriuretic peptides, and this fits with earlier reports that nitric oxide, which utilises cyclic GMP as a second messenger, prevents hypertrophy of neonatal and adult cardiomyocytes [4,29].

Many of the intracellular actions of cyclic GMP are mediated via activation of a cyclic GMP-dependent serine/threonine kinase [30]. The soluble isoform, cyclic GMP-dependent protein kinase-I, has been described in a number of vascular cell types and probably mediates cyclic GMP actions in the heart and vasculature [31]. Cyclic GMP-dependent protein kinase-I has been shown to mediate the antiproliferative effects of ANP in mesangial cells [32] and contribute to the antihypertrophic actions of an NO donor in neonatal cardiomyocytes [33]. In the present study, the antihypertrophic actions of BNP and CNP were completely abolished by an inhibitor of cyclic GMP-dependent protein kinase, KT5823 (Fig. 4B), highlighting the importance of cyclic GMP-dependent signalling in the cardiomyocyte responses to BNP and CNP. Cyclic GMP-dependent protein kinase inhibits signalling of key hypertrophic kinases in several cell types, particularly the extracellular signal regulated kinase (ERK1/2) pathway, and this may contribute to the present antihypertrophic action of the natriuretic peptides [32,34,35]. While the antihypertrophic effect of ANP has also been reported to require activation of ERK [36], that single study utilised sparsely plated neonatal rat cardiomyocytes, and the antihypertrophic actions described here are more likely to involve inhibition of hypertrophic MAPK signaling downstream of cyclic GMP-dependent protein kinase activation.

In contrast to ANP and BNP, CNP did not consistently increase levels of cyclic GMP (Fig. 3), despite all three natriuretic peptides exerting a similar antihypertrophic efficacy (Fig. 1). The rat heart expresses mRNA for all three natriuretic peptide receptors [9], but adult cardiomyocytes may have reduced or absent levels of functional NPR-B receptors [37]. However, CNP can still activate NPR-A [38]. In addition, the potent inhibition of the CNP response by KT5823 (Fig. 4B) highlights the essential nature of cyclic GMP-dependent signaling for the antihypertrophic actions of CNP in adult rat cardiomyocytes. These findings suggest that only small increases in cyclic GMP are sufficient to counter pro-hypertrophic stimuli in these cells, or that elevation of cyclic GMP is very compartmentalised.

Unlike BNP and CNP, the antihypertrophic response to ANP was not abolished by the cyclic GMP-dependent protein kinase inhibitor KT5823 (Fig. 4B), which suggests that ANP can activate additional, cyclic GMP-independent pathways. The signal transduction pathways by which ANP may prevent cellular growth are not well-defined, particularly in the cardiomyocyte. However ANP can prevent mitogen-activated protein kinase (MAPK) and protein kinase C signalling in vascular smooth muscle cells and astroglia, via both cyclic GMP-dependent and -independent mechanisms, including activation of MAPK phosphatase-1 [39] and activation of the clearance receptor NPR-C [40–43]. NPR-C may signal through inositol phosphates and inhibition of adenylyl cyclase, independently of cyclic GMP [38,44,45]. NPR-C is activated by all three natriuretic peptides, with binding affinities ANP>CNP>BNP [46], and might account for at least part of the response to ANP. In addition, ANP has been report to exert a modulatory action on ion channel [47,48] and phosphodiesterase activity [49]. Alternatively, ANP may elicit the observed effect via an as yet unidentified novel receptor. The antihypertrophic actions of all three natriuretic peptides were attenuated by HS-142-1, the dual NPR-A/NPR-B inhibitor. However the inhibition was more marked for BNP and CNP, roughly 2-fold that observed for ANP (Fig. 2B). This might reflect additional antihypertrophic activity of ANP independent of particulate guanylyl cyclase and its downstream cyclic GMP-dependent protein kinase, which would fit with the importance of ANP, rather than BNP, in regulating cardiac growth in vivo [11,13,14,25].

In conclusion, we demonstrate that the natriuretic peptides BNP and CNP share the direct antihypertrophic activity of ANP in adult rat cardiomyocytes. This novel finding is of interest particularly since BNP appears to exert antifibrotic rather than antihypertrophic actions in vivo [12], while CNP is thought to act at least in part via the endothelium [8]. Stimulation of a cyclic GMP-dependent pathway, linked to particulate guanlylyl cyclase activation, is common to the antihypertrophic mechanisms of all three natriuretic peptides in vitro. This had previously been demonstrated for ANP only in the immature cardiomyocyte. Interestingly, BNP and CNP act through the downstream cyclic GMP-dependent protein kinase, whereas ANP is able to prevent acute cardiomyocyte hypertrophy independently of this pathway.

Time for primary review 21 days.

	Acknowledgements

 
We thank Ms Lerna Gulluyan (Howard Florey Institute) for her invaluable advice regarding real time PCR. This study was supported by the High Blood Pressure Research Foundation of Australia, the National Health and Medical Research Council of Australia, and the Heart Foundation of Australia. Ms Rosenkranz was supported by an Australian Postgraduate Award.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 

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