Skip Navigation

Cardiovascular Research 2001 51(2):255-264; doi:10.1016/S0008-6363(01)00318-2
© 2001 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Autelitano, D. J
Right arrow Articles by Tang, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Autelitano, D. J
Right arrow Articles by Tang, F.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2000, European Society of Cardiology

Adrenomedullin is a regulated modulator of neonatal cardiomyocyte hypertrophy in vitro

Dominic J Autelitanoa,*, Rebecca Ridingsa and Fai Tangb

aMolecular Physiology Laboratory, Baker Medical Research Institute, P.O. Box 6492 St. Kilda Rd. Central, Melbourne, VIC 8008, Australia
bDepartment of Physiology, University of Hong Kong, Hong Kong, China

* Corresponding author. Tel.: +61-3-9522-4377; fax: +61-3-9521-1362 dominic.autelitano{at}baker.edu.au

Received 29 December 2000; accepted 9 April 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Adrenomedullin is a potent hypotensive, natriuretic and diuretic peptide that is coexpressed in the heart with its receptor, suggesting that it may have localized actions as a modulator of cardiac function. Although expression of adrenomedullin is upregulated in the pathological heart, its cardiac function has not been clearly elucidated and it is not known whether this represents a common feature of cardiac hypertrophy, nor whether this is restricted to cardiac myocytes. We have determined the direct effects of hypertrophic agents on cardiomyocyte adrenomedullin gene expression and peptide secretion and have examined the effects of adrenomedullin on biochemical markers of cardiomyocyte hypertrophy. Methods: Regulation of adrenomedullin expression and its effects on the hypertrophic response were studied in cultured rat neonatal ventricular cardiomyocytes. Results: Incubation with phenylephrine or endothelin for 48 h led to a hypertrophic response with an associated fivefold stimulation of ANP gene expression. In contrast, adrenomedullin mRNA was inhibited by 30–50% in response to phenylephrine or endothelin-mediated hypertrophy, and this was associated with a 35–45% reduction in secretion of immunoreactive adrenomedullin. Phorbol ester mediated activation of protein kinase C and increasing intracellular Ca2+ with ionomycin led to significant downregulation of adrenomedullin gene expression in cardiomyocytes. Co-incubation with 100 nM adrenomedullin for 48 h inhibited phenylephrine-induced cardiomyocyte hypertrophy as determined by protein:DNA ratio. Adrenomedullin partially blocked phenylephrine-mediated transcriptional activation of ANP and MLC-2 reporter gene expression in cardiomyocytes and this effect was mimicked by 2 µM forskolin, suggesting that this response was mediated via the activation of adenylate cyclase. Conclusion: These data demonstrate that the cardiomyocyte adrenomedullin gene is repressed by phenylephrine or endothelin-mediated hypertrophy. The inhibitory effects of adrenomedullin on the cardiomyocyte hypertrophic response suggests that this peptide acts as a regulated autocrine or paracrine modulator of cardiomyocyte function and that downregulation of adrenomedullin expression may play a role in induction and maintenance of cardiomyocyte hypertrophy.

KEYWORDS Adrenergic (ant)agonists; Endothelins; Gene expression; Hypertrophy; Receptors


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Adrenomedullin (AM) is a vasoactive peptide hormone that has been shown to have potent hypotensive actions in a variety of species [1]. In addition to its vascular effects, administration of AM has natriuretic and diuretic actions [1], as well as direct actions on the heart [2], suggesting that AM represents an important hormone involved in regulation of cardiovascular physiology. Immunoreactive (ir)-AM has been found in human plasma, with significantly elevated levels that increase in proportion to clinical severity in hypertensive [3] and congestive heart failure patients [4,5]. Immunocytochemical staining of human heart tissue showed that ir-AM is expressed in cardiomyocytes [5] and in addition, ir-AM has been shown to be released into the coronary sinus in response to myocardial ischemia, suggesting that such damage results in release of AM from the heart [6]. High expression of AM mRNA and ir-AM has also been demonstrated in vascular endothelium and smooth muscle cells [1], indicating that cells other than cardiomyocytes may produce AM in the heart, and raising the possibility that these non-myocardial cells may additionally contribute to the overall cardiac expression of AM. Animal models have demonstrated that expression of AM can be upregulated in the heart in some models of hypertension, cardiac hypertrophy and cardiac ischemia. Aortocaval shunt-induced volume overload caused sustained elevation of AM mRNA and ir-AM peptide levels in rat LV [7], while pressure overload hypertrophy induced by monocrotaline [7] or aortic banding [8] had no effect on LV AM mRNA. Experimental ischemic heart failure in rats also causes a marked and prolonged upregulation of AM expression, both within and outside the infarcted region of the myocardium [9].

A number of studies have indicated that factors such as angiotensin II, endothelin, and insulin-like growth factor can be activated within the heart and are likely to play an autocrine/paracrine role in the establishment of cardiomyocyte hypertrophy and progression to heart failure [10–12]. Adrenomedullin binding sites are abundant in heart [13], and we have previously shown that adrenomedullin receptor genes are expressed in cardiac myocytes [14]. Taken together, the localised expression of adrenomedullin and its receptor strongly suggest that this peptide may function as an intracardiac regulator of myocyte function. This notion is supported by studies demonstrating that exogenously administered AM inhibits angiotensin II-stimulated [14C]phenylalanine incorporation in neonatal cardiomyocyte cultures, and in addition, blocking endogenous AM action with a monoclonal anti-AM antibody enhances the effects of angiotensin II [15].

Despite in vivo evidence that AM can be upregulated during cardiac hypertrophy and the progression to heart failure, whether or not these changes are specific to the cardiomyocyte and the mechanisms involved in regulating its expression are not known. In the present study, we have used cultured neonatal cardiomyocytes to directly determine whether AM expression is regulated by agents known to induce a hypertrophic phenotype and have also determined the effects of exogenous AM on the cardiomyocyte hypertrophic growth response and on expression of hypertrophy-induced genes. The current data demonstrate that AM has anti-hypertrophic effects and that cardiomyocyte AM gene expression is downregulated in response to phenylephrine or endothelin, suggesting that repression of local AM production may play a role in the maintenance of the hypertrophic phenotype in the cardiac myocyte.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Neonatal cardiomyocyte cultures
Primary cultures of cardiac myocytes (MC) were prepared from 1–2-day-old Sprague–Dawley rat pups in accordance with the Australian Code of Practise for the Care and Use of Animals for Scientific Purposes as described [16]. Following initial digestion with 0.1% collagenase for 15 min at 37°C, ventricles were washed in dissociation buffer (calcium-free Dulbecco's phosphate buffered saline containing 1 mM MgSO4) and single cell preparations were then prepared by multiple 5-min incubations with 0.08% trypsin and 0.01% DNase. Cells were then pre-plated in 100-mm culture dishes at 37°C for 30 min to remove the majority of fibroblasts. MC were removed, counted and plated at an initial density of 750 cells/mm2 for 18 h in DMEM containing 10% FCS+5% horse serum, 0.1 mM bromodeoxyuridine (BdU), and penicillin-streptomycin-fungizone solution (1:100 dilution, CSL, Parkville, VIC, Australia). MC were then washed in serum-free DMEM containing 0.1 mM BdU, 10 µg/ml bovine transferrin (Sigma) and 2.5 U/ml human insulin (Novo Nordisk Pharmaceuticals, Australia), incubated in this medium for a further 48 h, and experiments were performed by incubating cells in the same medium without BdU. Phenylephrine-hydrochloride (PE) and propranololol-hydrochloride (PRO) were purchased from Sigma Chemicals and were used at final concentrations of 25 µM and 1 µM, respectively. In all experiments using PE, PRO was included to block β-adrenergic receptors. Human endothelin-1 (ET) was obtained from Auspep (Parkville, VIC, Australia) and rat adrenomedullin was purchased from Phoenix Pharmaceuticals (Belmont, CA, USA) and were used in culture experiments at the concentrations indicated. Phorbol-12-myristate-13-acetate (PMA) and ionomycin were both purchased from Calbiochem and were dissolved in DMSO.

2.2 RNA preparation and ribonuclease protection analysis
Following incubation with test substances, medium was aspirated from MC cultures, the cells washed with phosphate buffered saline and total RNA isolated as previously described [16].

A 613-nt RT-PCR cloned rat AM cDNA fragment encompassing the entire coding region, a 755-nt rat ANP cDNA and a 177-nt rat GAPDH cDNA clone (GAPDH-177) were used as templates to generate 32P-labeled cRNA probes for use in solution hybridization/RNase protection analysis as previously described [16,17]. AM and ANP probes were labelled to a specific activity of ~5x108 cpm/µg RNA and GAPDH probes to 2x108 cpm/µg RNA. Solution hybridization and RNase digestion was carried out as described previously [16,17] and protected RNA hybrids were analyzed on non-denaturing polyacrylamide gels and quantitated on a Fuji BAS-1000 phosphorimaging system. Quantitation of ANP and AM mRNA transcripts was achieved by comparison to standard curves generated following RNase protection of known amounts of in vitro synthesized sense RNA transcripts as described [16]. Levels of GAPDH mRNA, simultaneously hybridized in each sample, were used to correct for minor differences in RNA loading.

2.3 Transient transfection and reporter gene assays
For transient transfection, MC in 12-well plates were washed 24 h post-plating with DMEM with no additives and then transfected with 0.5 µg DNA and 1.5 µl Tfx-50 (Promega) in 400 µl DMEM for 1 h. Transfection medium was overlaid with 1 ml DMEM containing 0.1 mM BdU, 10 µg/ml bovine transferrin and 2.5 U/ml human insulin and MC were incubated for a further 24 h. Medium was then replaced with DMEM containing 10 µg/ml bovine transferrin, 2.5 U/ml human insulin and the appropriate test substances as indicated. Transcriptional activity of the ANP and myosin light chain 2 (MLC-2) promoters were measured using a plasmid containing a 700 base-pair fragment of the rat ANP promoter linked to firefly luciferase (ANP328 [18], courtesy of Dr. M. Nema) or the MLC-2 promoter linked to firefly luciferase ([19]; courtesy of Dr. J. Heller Brown). Luciferase activity was measured in 20 µl aliquots after 24–48 h treatment following lysis in 200 µl reporter lysis buffer (Promega).

2.4 Radioimmunoassay of AM
Media samples were extracted on Sep-Pak C18 columns (Waters, MA, USA) with 80% methanol+0.1% trifluoroacetic acid as eluting solvent and peptide extracts were lyophilized and reconstituted in radioimmunoassay buffer for determination of immunoreactive (ir)-AM concentration as described previously [17]. The DNA content of each well was determined [20] as an indicator of MC number in each experiment and was used to normalize ir-AM secretion data.

2.5 Statistical analysis
For all multi-group comparisons, data were analyzed by one-way ANOVA followed by Fisher's PLSD for post hoc comparisons. Student's t-test was used for comparisons between two groups. All data are presented as mean±S.E.M. and P<0.05 was considered to be significantly different.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Cardiomyocyte hypertrophy in vitro inhibits AM gene expression and peptide release
To directly determine the influence of hypertrophy on cardiomyocyte AM expression, MC cultures were treated for 48 h with the alpha adrenergic ({alpha}-AR) receptor agonist phenylephrine (PE) or endothelin-1 (ET), which are well recognised stimulators of cardiomyocyte hypertrophy [21]. MC maintained in defined serum free did not hypertrophy, but were readily responsive to various hypertrophic agents as previously shown [16]. After 48 h incubation with 25 µM PE or 50 nM ET, cardiomyocytes displayed an increase in cell size, had made extensive cell–cell contacts, and were beating synchronously. Associated with these changes in cellular morphology was a significant increase in cellular protein and total RNA, indicative of a hypertrophic response (data not shown).

In addition to changes in cellular morphology and increased biosynthesis of both RNA and protein, levels of ANP mRNA were also assessed in MC cultures as an indicator of cardiomyocyte hypertrophy (Fig. 1B). In ventricular MC cultures incubated in the presence of PE or ET for 48 h, ANP mRNA was elevated 4–5-fold above control levels (Con, 26±8; PE, 113±6; ET, 130±5 amol/µg RNA).


Figure 1
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Solution hybridization/RNase protection of AM, ANP and GAPDH mRNA following in vitro induction of hypertrophy in neonatal ventricular cardiomyocytes. Cardiomyocyte cultures were treated for 48 h with 25 µM PE+1 µM PRO or 50 nM ET and total RNA was used for simultaneous hybridization with either (A) GAPDH and a probe spanning the entire AM prohormone coding region (2 µg RNA) or (B) ANP/GAPDH probes (0.2 µg RNA). Phosphorimages shown represent RNase protected bands from individual cultures. Quantitative data represents mean±S.E.M. of 7–8 cultures. *P<0.01 versus control. (C) RNase protection of a 225-nt fragment of exon 4 of rat AM mRNA following 48 h PE-induced hypertrophy. (D) RT-PCR of a 613-nt fragment of AM mRNA spanning exons 1–4 or a 402-nt fragment of ANP mRNA from control or 48 h PE-treated cardiomyocyte RNA.

 
In sharp contrast to ANP, RNase protection analysis of AM mRNA using a probe that spans part of exon 1 and the entire AM prohormone coding region (all of exons 2, 3 and part of exon 4) demonstrated that AM mRNA expression is downregulated as part of the hypertrophic phenotype in the cardiomyocyte (Fig. 1A). Both PE and ET caused significant inhibition of AM mRNA expression (Con, 1.3±0.14; PE, 0.87±0.06; ET, 0.43±0.05 amol/µg RNA, mean±S.E.M. of 7–8 cultures) in MC cultures treated for 48 h (Fig. 1A). Using a different AM mRNA probe complimentary to 225 nucleotides of exon 4 only, it was similarly shown that AM mRNA was downregulated ~5-fold in hypertrophied MC following 48 h stimulation with PE (Fig. 1C). Furthermore, RT-PCR using primers spanning exon 1–exon 4 of the rat AM sequence confirmed that PE led to downregulation of full length AM mRNA transcripts, while ANP primers demonstrated upregulated ANP mRNA in the same samples (Fig. 1D).

Release of ir-AM from cardiomyocyte cultures was measured by specific radioimmunoassay following 48 h incubation with or without hypertrophic agents. Control cardiomyocytes actively secreted ir-AM into the medium (116±22 pg/µg DNA over a 48 h period), and peptide release was significantly inhibited in MC that had been incubated in the presence of either PE or ET (Table 1). Accumulated ir-AM secreted over a 48 h period was 35–45% lower (64±2 and 74±6 pg/µg DNA) in PE or ET treated cells, respectively, in comparison to control MC.


View this table:
[in this window]
[in a new window]

  		Table 1 Release of immunoreactive AM from neonatal ventricular cardiomyocyte cultures

 
3.2 Decay of AM mRNA in control versus hypertrophied cardiomyocytes
To test whether the downregulation of AM mRNA in hypertrophied MC may be due to alterations in transcript stability potentially related to AU rich motifs in the 3' tail of the AM mRNA, MC were treated for 48 h with PE after which actinomycin D (2 µg/ml) was added to the cultures to arrest gene transcription. Prior to the addition of actinomycin D, AM mRNA expression was downregulated to ~15% of control levels in MC treated with PE for 48 h (Fig. 2). Although the level of GAPDH mRNA expression did not change after acute transcriptional arrest with actinomycin D, AM mRNA decayed rapidly in control MC, with an estimated half-life of 53 min. Despite the attenuated expression in PE-treated MC, AM mRNA decayed in a similar fashion to control cells, with an estimated half-life of 61 min (Fig. 2).


Figure 2
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Decay of AM mRNA in control and hypertrophied neonatal cardiomyocyte cultures. MC cultures were treated in the presence or absence of 25 µM PE+1 µM PRO for 48 h and actinomycin D (2 µg/ml) was then added for the indicated times to arrest gene transcription. Total RNA was harvested from each well and the entire amount (one well equivalent) was used for simultaneous solution hybridization/RNase protection of AM and GAPDH mRNA. (A) Representative RNase protected bands showing changes in AM and GAPDH mRNA, and (B) quantitative changes in AM and GAPDH mRNA transcripts. Data represent mean±S.E.M. of three individual cultures.

 
3.3 Protein kinase C and intracellular Ca2+ inhibit AM mRNA expression
Both protein kinase C (PKC) and intracellular Ca2+ have been reported to play crucial roles in the development of cardiomyocyte hypertrophy and are likely to be involved in the response to {alpha}-AR and ET-1 receptor activation [21]. We therefore determined whether activation of PKC or elevation of intracellular Ca2+ could directly mediate suppression of AM mRNA levels. Following 18 h incubation with 100 nM PMA, cardiomyocytes appeared larger in size, had established numerous cell–cell contacts and were spontaneously beating. Associated with this PMA-induced hypertrophic response, AM mRNA expression was reduced by 50% (P<0.001) relative to control cells (Fig. 3A). Cardiomyocytes incubated with the Ca2+ ionophore ionomycin (500 nM) for 18 h displayed increased spontaneous contractions and a small but significant (20% lower than control; P<0.01) decrease in AM mRNA transcript levels (Fig. 3A). As an internal control, expression of ANP mRNA was determined in RNA extracted from the same cultures. PMA led to a significant fivefold increase in ANP mRNA levels while the Ca2+ ionophore led to a small twofold increase in the mean level of ANP mRNA that did not reach statistical significance (Fig. 3B).


Figure 3
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Activation of protein kinase C and elevation of intracellular Ca2+ inhibit adrenomedullin gene expression in neonatal ventricular cardiomyocytes. Cardiomyocyte cultures were treated with 100 nM PMA or 500 nM ionomycin for 18 h and RNA was analysed by solution hybridization/RNase protection for expression of (A) AM and GAPDH mRNA (3 µg RNA hybridized) or (B) ANP mRNA transcripts (0.5 µg RNA hybridized). Left panels show representative changes following polyacrylamide gel electrophoresis and phosphorimage analysis and right hand panels show quantitative analysis (mean±S.E.M. of four individual cultures; *P<0.01 versus control).

 
3.4 AM inhibits cardiomyocyte hypertrophy and transcriptional activity of MLC-2 and ANP promoters
We have previously demonstrated that cardiac myocytes express receptors for AM [14], suggesting that locally secreted AM may play an autocrine or paracrine role in regulating cardiomyocyte function. We therefore determined the effects of exogenously administered AM on various parameters of the cardiomyocyte hypertrophic response. The overall hypertrophic growth response of cardiomyocytes was determined by measuring protein:DNA ratio 48 h post-treatment with PE in the absence or presence of AM (Fig. 4A). Although AM alone had no significant effect, PE-mediated cardiomyocyte hypertrophy was characterised by a 1.5-fold increase (P=0.02) in protein:DNA ratio. In the presence of 100 nM AM however, PE failed to cause a significant hypertrophic response in cardiomyocyte cultures as measured by protein:DNA ratio (Con, 5.0±0.7; PE, 7.90±1.1; AM, 4.3±0.25; AM+PE, 5.7±0.35: mean±S.E.M., n=4).


Figure 4
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 AM inhibits cardiomyocyte hypertrophy and transcriptional activation of hypertrophy response genes. (A) Cardiomyocytes were treated in the presence of 25 µM PE+1 µM PRO, 100 nM AM or a combination of both for 48 h. Cytoplasmic protein content was measured and expressed relative to DNA recovered from each culture. Data shown represents mean±S.E.M. of four individual cultures; *P<0.05; NS, not significantly different. Activity of MLC-2 (B) and ANP (C) promoters in cardiomyocyte cultures following 48 h incubation in the presence of 25 µM PE+1 µM PRO, 100 nM AM or a combination of both. Data shown represents mean±S.E.M. of 4–8 individual cultures; *P<0.02 compared with PE alone. (D) ANP promoter activity following 24 h stimulation with 25 µM PE+1 µM PRO in the presence or absence of 100 nM AM, 0.1 µM or 2 µM forskolin (FSK). Data shown represents mean±S.E.M. of four individual cultures; *P<0.02 versus PE-treated group.

 
To determine whether AM influences the transcriptional activity of genes known to be activated as part of the hypertrophic phenotype, we examined the effects of AM on the PE-stimulated activity of luciferase reporters under the control of either the MLC-2 or ANP promoters. Basal transcriptional activity of the MLC-2 or the ANP promoter constructs was not significantly altered after 48 h incubation in the presence of 100 nM AM (Fig. 4B and C). PE alone led to significant transcriptional stimulation of the MLC-2 (7.4-fold above control, P<0.0001) and ANP (2.9-fold above control, P<0.0001) promoters. In the presence of 100 nM AM, the stimulatory effect of PE on both MLC-2 and ANP reporter gene transcription was partially blocked relative to the effects of PE alone. AM inhibited PE-induced transcription of the MLC-2 and ANP gene promoters by ~40% and 25%, respectively (Fig. 4B and C).

We further determined whether the inhibitory effects of AM on PE-stimulated ANP gene transcription could be mimicked by the adenylate cyclase activator forskolin. Incubation with PE (24 h) led to an eightfold increase in ANP-Luc activity which was inhibited by ~40% in the presence of 100 nM AM (Fig. 4D). Coincubation of cardiomyocyte cultures with low dose forskolin (0.1 µM) had no effect, however, 2 µM forskolin blocked PE-stimulated ANP-Luc activity to a similar extent as 100 nM AM suggesting that the inhibitory effects of AM may be mediated via the activation of cAMP.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Since its discovery in 1993, AM has been shown to have a range of biological effects including vasodilation, natriuresis and diuresis [1], suggesting that AM may play a similar role to ANP in regulating cardiovascular responses. The sustained upregulation of ANP gene expression in ventricular cardiomyocytes is a consistent marker of hypertrophy in response to a wide variety of agents both in vivo and in vitro [21]. In the present study, we have shown that AM mRNA expression is downregulated in hypertrophied cardiomyocytes, suggesting that these two genes are differentially expressed and regulated in cardiomyocytes. The relatively low level of cardiac expression of AM and the colocalization of AM receptors in cardiomyocytes [14] is consistent with this peptide having localised autocrine/paracrine effects.

Both {alpha}-AR and ET-1 receptors mediate hypertrophic signalling in cardiomyocytes via coupling to G{alpha}q which in turn leads to PKC activation, alterations in Ca2+ signalling and activation of mitogen activated protein kinase (MAPK) pathways [22]. Direct activation of PKC with PMA leads to hypertrophic growth of cardiomyocytes [23] associated with transcriptional activation of ANP and MLC-2 gene promoters [19], suggesting that PKC is a key mediator of the cardiomyocyte hypertrophic response. In our studies, activation of {alpha}-AR or ET-1 receptors led to an obvious hypertrophic response in cardiomyocytes that was characterised by induction of total cellular RNA and protein and MLC-2 and ANP gene transcription. In marked contrast to MLC-2 and ANP, however, the hypertrophic response to ET or PE in cultured MC caused significant downregulation of AM mRNA and ir-AM secretion. The phorbol ester PMA and the Ca2+ ionophore ionomycin similarly had opposite effects on ANP and AM gene expression in cardiomyocyte cultures, raising the possibility that PKC and intracellular Ca2+ are key mediators of the hypertrophy-mediated repression of AM gene expression. A diverse range of genes have been shown to be upregulated in the hypertrophied heart [24], however, relatively few downregulated genes have been described. Like AM, the cardiomyocyte sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene has been shown to be downregulated by hypertrophic agents such as endothelin in a PKC-dependent manner [25,26], suggesting that the AM and SERCA2 genes may be repressed during cardiomyocyte hypertrophy in a similar manner. Although the precise molecular mechanisms have not been fully elucidated, there is evidence to suggest that PMA-induced mRNA destabilization may in part be responsible for SERCA2 mRNA downregulation [26]. AM mRNA transcripts contain multiple AU-rich sequences that may be involved in mRNA destabilization, however, AM mRNA half-life was unchanged following PE-induced hypertrophy, suggesting that transcriptional repression may be the major mechanism involved in downregulating AM mRNA levels. In mouse C3H10T1/2 fibroblasts, transcription of the AM gene is downregulated by Myc-mediated repression via an initiator element (INR) in the AM promoter [27]. Since c-myc gene expression is rapidly induced in cardiomyocytes in response to {alpha}-AR activation [28], it is possible that this might represent a mechanism for transcriptional repression of the AM gene.

Despite the clear inhibitory effects of hypertrophy on neonatal cardiomyocyte AM expression in vitro, in vivo studies in adult rats show differential effects on cardiac AM expression. Whether this reflects differences between neonatal and adult cardiomyocytes or merely the cumulative measurement of AM expression in multiple cell types in whole heart is not clear. Volume overload induced by aortocaval shunt causes sustained elevation of AM mRNA and ir-AM peptide levels in rat LV [7], while monocrotaline-induced pressure overload hypertrophy [7] or aortic banding [8] had no effect on LV AM mRNA. Taken together, these data suggest that the cardiac AM gene is not upregulated as part of the hypertrophic phenotype per se and that its induction is dependent upon the etiology of the hypertrophy. Consistent with this, it was shown that acute pressure overload produced in rats by infusion of arginine vasopressin for 2 h caused increased ventricular expression of AM mRNA that was independent of myocyte hypertrophy [29]. On the other hand, ventricular AM gene expression is consistently induced in the failing heart [5,8,9,30], suggesting AM gene activation as ventricular dysfunction progresses. Conflicting data have been presented regarding the precise cellular localization of these changes however, with increased immunocytochemical staining of ir-AM shown in cardiac myocytes [5] and microvascular endothelial cells [9] of the failing heart. Thus it is likely that AM expression may be regulated in multiple cell populations in the pathological heart, and that overall cardiac AM levels do not necessarily reflect expression in cardiac myocytes.

The heart has a high density of AM binding sites [13] and putative AM receptor genes are expressed in cardiomyocytes [14], implying that these cells are direct targets of AM action. Consistent with a previous study demonstrating that AM inhibits angiotensin II-stimulated [14C]phenylalanine incorporation in neonatal cardiomyocytes [15], we have shown that AM can block PE-mediated cardiomyocyte hypertrophy as determined by inhibition of increases in cellular protein:DNA ratio. In addition to its effects on cellular hypertrophy, AM partially blocked the PE-stimulated transcription of two genes (ANP and MLC-2) that are well-established markers of the cardiomyocyte hypertrophic phenotype [21].

The dose-dependent accumulation of cAMP in cardiomyocytes in response to AM has been described [31,32], suggesting that elevated levels of cAMP may play a role in the antihypertrophic effects of AM. Consistent with this, receptor-independent activation of adenylate cyclase with 2 µM forskolin led to similar inhibitory effects on PE-stimulated ANP gene transcription. Although the present data provide evidence for AM-mediated inhibitory effects on several parameters of the cardiomyocyte hypertrophic response that may be dependent upon increased cAMP accumulation, the molecular mechanisms involved are not known. Hypertrophic signalling in cardiomyocytes appears to involve Ras-dependent activation of Raf-1 which in turn leads to activation of MAPK pathways [21]. An inhibitory role for cAMP-dependent protein kinase A has been demonstrated in several cell types that effectively blocks Ras-dependent activation of Raf-1 and subsequent activation of downstream MAPK [33]. Though not established in the cardiomyocyte, inhibitory PKA-dependent phopshorylation of Raf-1 could account for some of the antihypertrophic effects of increased intracellular cAMP. In addition, it has been proposed that cAMP may have either positive or negative effects on cardiomyocyte ANP secretion that are related to its effects on Ca2+ transients and cellular contractile state [34]. While low doses of forskolin (0.1 µM) were shown to increase cardiomyocyte Ca2+ transients, contractile frequency and ANP secretion, higher doses of forskolin inhibited these parameters [34], implying that the absolute level of cAMP may be a crucial determinant of the cellular response. Since cellular contraction is a potent stimulus for cardiomyocyte hypertrophy [23], it is possible that the antihypertrophic effects of AM and 2 µM forskolin seen in the current study may be due to subtle alterations in the contractile state of the cardiomyocyte cultures. The differences in previously demonstrated direct cAMP-dependent [35] and cAMP-independent [36] positive inotropic actions, as well as negative inotropic effects of AM [37] may also relate to the concentration of AM used.

In summary, the present study demonstrates that in marked contrast to the ANP gene, induction of cardiomyocyte AM expression is not a general feature of hypertrophy, and that in vitro, hypertrophy induced by the activation of G{alpha}q-coupled receptors ({alpha}1-AR and ET-1) leads to significant repression of cardiomyocyte AM gene expression and peptide secretion. The inhibitory actions of AM on the cardiomyocyte hypertrophic response suggest that AM acts as an intracardiac modulator of cardiomyocyte function and that repression of AM gene expression may act in part to maintain hypertrophic signalling.

Time for primary review 31 days.


   Acknowledgements
 
We thank Brian Jones for digital imaging expertise and preparation of figures. Supported in part by a grant in aid to DJA from the National Heart Foundation of Australia.


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

  1. Hinson J.P., Kapas S., Smith D.M. Adrenomedullin, a multifunctional regulatory peptide. Endocr Rev (2000) 21:138–167.[Abstract/Free Full Text]
  2. Parkes D.G., May C.N. Direct cardiac and vascular actions of adrenomedullin in conscious sheep. Br J Pharmacol (1997) 120:1179–1185.[CrossRef][Web of Science][Medline]
  3. Kitamura K., Ichiki Y., Tanaka M., et al. Immunoreactive adrenomedullin in human plasma. FEBS Lett (1994) 341:288–290.[CrossRef][Web of Science][Medline]
  4. Kato J., Kobayashi T., Etoh T., et al. Plasma adrenomedullin concentration in patients with heart failure. J Clin Endocrinol Metab (1996) 81:180–183.[Abstract]
  5. Jougasaki M., Wei C.-M., McKinley L.J., Burnett J.C. Jr. Elevation of circulating and ventricular adrenomedullin in human congestive heart failure. Circulation (1995) 92:286–289.[Abstract/Free Full Text]
  6. Vijay P., Szekely L., Aufiero T.X., Sharp T.G. Coronary sinus adrenomedullin rises in response to myocardial injury. Clin Sci (1999) 96:415–420.[CrossRef][Web of Science][Medline]
  7. Yoshihara F., Nishikimi T., Horio T., et al. Ventricular adrenomedullin concentration is a sensitive biochemical marker for volume and pressure overload in rats. Am J Physiol Heart Circ Physiol (2000) 278:H633–642.[Abstract/Free Full Text]
  8. Kaiser M., Kahr O., Shimada Y., et al. Differential regulation of ventricular adrenomedullin and atrial natriuretic peptide gene expression in pressure and volume overload in the rat. Clin Sci (1998) 94:359–365.[Web of Science][Medline]
  9. Oie E., Vinge L.E., Yndestad A., et al. Induction of a myocardial adrenomedullin signaling system during ischemic heart failure in rats. Circulation (2000) 101:415–422.[Abstract/Free Full Text]
  10. Ito H., Hirata Y., Adachi S., et al. Endothelin-1 is an autocrine/paracrine factor in the mechanism of angiotensin II-induced hypertrophy in cultured rat cardiomyocytes. J Clin Invest (1993) 92:398–403.[Web of Science][Medline]
  11. Sadoshima J., Xu Y., Slayter H.S., Izumo S. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell (1993) 75:977–984.[CrossRef][Web of Science][Medline]
  12. Serneri G.G., Modesti P.A., Boddi M., et al. Cardiac growth factors in human hypertrophy. Relations with myocardial contractility and wall stress. Circ Res (1999) 85:57–67.[Abstract/Free Full Text]
  13. Owji A.A., Smith D.M., Coppock H.A., et al. An abundant and specific binding site for the novel vasodilator adrenomedullin in the rat. Endocrinology (1995) 136:2127–2134.[Abstract]
  14. Autelitano D.J. Cardiac expression of genes encoding putative adrenomedullin/calcitonin gene-related peptide receptors. Biochem Biophys Res Commun (1998) 250:689–693.[CrossRef][Web of Science][Medline]
  15. Tsuruda T., Kato J., Kitamura K., et al. Adrenomedullin: a possible autocrine or paracrine inhibitor of hypertrophy of cardiomyocytes. Hypertension (1998) 31:505–510.[Abstract/Free Full Text]
  16. Autelitano D.J., Woodcock E.A. Selective activation of alpha1A-adrenergic receptors in neonatal cardiac myocytes is sufficient to cause hypertrophy and differential regulation of alpha1-adrenergic receptor subtype mRNAs. J Mol Cell Cardiol (1998) 30:1515–1523.[CrossRef][Web of Science][Medline]
  17. Autelitano D.J., Tang F., Little P.J. Rapid regulation of adrenomedullin in metabolically compromised vascular smooth muscle cells. J Hypertens (1999) 17:373–379.[CrossRef][Web of Science][Medline]
  18. Argentin S., Sun Y.L., Lihrmann I., et al. Distal cis-acting promoter sequences mediate glucocorticoid stimulation of cardiac atrial natriuretic factor gene transcription. J Biol Chem (1991) 266:23315–23322.[Abstract/Free Full Text]
  19. Shubeita H.E., Martinson E.A., Van Bilsen M., Chien K.R., Brown J.H. Transcriptional activation of the cardiac myosin light chain 2 and atrial natriuretic factor genes by protein kinase C in neonatal rat ventricular myocytes. Proc Natl Acad Sci USA (1992) 89:1305–1309.[Abstract/Free Full Text]
  20. Burton K. A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J (1955) 62:315–323.[Web of Science]
  21. Hefti M.A., Harder B.A., Eppenberger H.M., Schaub M.C. Signaling pathways in cardiac myocyte hypertrophy. J Mol Cell Cardiol (1997) 29:2873–2892.[CrossRef][Web of Science][Medline]
  22. Dorn G.W. II, Brown J.H. Gq signaling in cardiac adaptation and maladaptation. Trends Cardiovasc Med (1999) 9:26–34.[CrossRef][Web of Science][Medline]
  23. Allo S.N., Carl L.L., Morgan H.E. Acceleration of growth of cultured cardiomyocytes and translocation of protein kinase C. Am J Physiol (1992) 263:C319–325.[Web of Science][Medline]
  24. Johnatty S.E., Dyck J.R., Michael L.H., Olson E.N., Abdellatif M. Identification of genes regulated during mechanical load-induced cardiac hypertrophy. J Mol Cell Cardiol (2000) 32:805–815.[CrossRef][Web of Science][Medline]
  25. Hartong R., Villarreal F.J., Giordano F., et al. Phorbol myristate acetate-induced hypertrophy of neonatal rat cardiac myocytes is associated with decreased sarcoplasmic reticulum Ca2+ ATPase (SERCA2) gene expression and calcium reuptake. J Mol Cell Cardiol (1996) 28:2467–2477.[CrossRef][Web of Science][Medline]
  26. Qi M., Bassani J.W., Bers D.M., Samarel A.M. Phorbol 12-myristate 13-acetate alters SR Ca(2+)-ATPase gene expression in cultured neonatal rat heart cells. Am J Physiol (1996) 271:H1031–1039.[Medline]
  27. Wang X., Peters M.A., Utama F.E., Wang Y., Taparowsky E.J. The adrenomedullin gene is a target for negative regulation by the Myc transcription complex. Mol Endocrinol (1999) 13:254–267.[Abstract/Free Full Text]
  28. Starksen N.F., Simpson P.C., Bishopric N., et al. Cardiac myocyte hypertrophy is associated with c-myc protooncogene expression. Proc Natl Acad Sci USA (1986) 83:8348–8350.[Abstract/Free Full Text]
  29. Romppanen H., Marttila M., Magga J., et al. Adrenomedullin gene expression in the rat heart is stimulated by acute pressure overload: blunted effect in experimental hypertension. Endocrinology (1997) 138:2626–2639.
  30. Nishikimi T., Horio T., Sasaki T., et al. Cardiac production and secretion of adrenomedullin are increased in heart failure. Hypertension (1997) 30:1369–1375.[Abstract/Free Full Text]
  31. Sato A., Canny B.J., Autelitano D.J. Adrenomedullin stimulates cAMP accumulation and inhibits atrial natriuretic peptide gene expression in cardiomyocytes. Biochem Biophys Res Commun (1997) 230:311–314.[CrossRef][Web of Science][Medline]
  32. Ikeda U., Kanbe T., Kawahara Y., Yokoyama M., Shimada K. Adrenomedullin augments inducible nitric oxide synthase expression in cytokine-stimulated cardiac myocytes. Circulation (1996) 94:2560–2565.[Abstract/Free Full Text]
  33. Marx J. Two major signal pathways linked. Science (1993) 262:988–990.[Free Full Text]
  34. Church D.J., Rebsamen M.C., Morabito D., van Der Bent V., Vallotton M.B., Lang U. Role of cell contractions in cAMP-induced cardiomyocyte atrial natriuretic peptide release. Am J Physiol Heart Circ Physiol (2000) 278:H117–125.[Abstract/Free Full Text]
  35. Ihara T., Ikeda U., Tate Y., Ishibashi S., Shimada K. Positive inotropic effects of adrenomedullin on rat papillary muscle. Eur J Pharmacol (2000) 390:167–172.[CrossRef][Web of Science][Medline]
  36. Szokodi I., Kinnunen P., Tavi P., Weckstrom M., Toth M., Ruskoaho H. Evidence for cAMP-independent mechanisms mediating the effects of adrenomedullin, a new inotropic peptide. Circulation (1998) 97:1062–1070.[Abstract/Free Full Text]
  37. Ikenouchi H., Kangawa K., Matsuo H., Hirata Y. Negative inotropic effect of adrenomedullin in isolated adult rabbit cardiac ventricular myocytes. Circulation (1997) 95:2318–2324.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
K.-D. Schluter and K. C Wollert
Synchronization and integration of multiple hypertrophic pathways in the heart
Cardiovasc Res, August 15, 2004; 63(3): 367 - 372.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. M. White, P. E. Constantin, and W. C. Claycomb
Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function
Am J Physiol Heart Circ Physiol, March 1, 2004; 286(3): H823 - H829.
[Abstract] [Full Text] [PDF]

Home page
 J CARDIOVASC PHARMACOL THERHome page
J. Varagic, D. Susic, M. Slama, and E. D. Frohlich
Omapatrilat Induces Profound Renal Vasodilation but Does Not Affect Coronary Hemodynamics
Journal of Cardiovascular Pharmacology and Therapeutics, June 1, 2003; 8(2): 167 - 174.
[Abstract] [PDF]

Home page
 Cardiovasc ResHome page
R. Nakamura, J. Kato, K. Kitamura, H. Onitsuka, T. Imamura, K. Marutsuka, Y. Asada, K. Kangawa, and T. Eto
Beneficial effects of adrenomedullin on left ventricular remodeling after myocardial infarction in rats
Cardiovasc Res, December 1, 2002; 56(3): 373 - 380.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Autelitano, D. J
Right arrow Articles by Tang, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Autelitano, D. J
Right arrow Articles by Tang, F.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?