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(Circulation Research. 2003;93:246.)
© 2003 American Heart Association, Inc.

Integrative Physiology

Direct Actions of Urotensin II on the Heart

Implications for Cardiac Fibrosis and Hypertrophy

Alex Tzanidis, Ross D. Hannan, Walter G. Thomas, Döne Onan, Dominic J. Autelitano, Fiona See, Darren J. Kelly, Richard E. Gilbert, Henry Krum

From NHMRC Centre of Clinical Research Excellence in Therapeutics, Department of Medicine/Epidemiology and Preventive Medicine (A.T., H.K.), Monash University Medical School, and Baker Medical Research Institute (R.D.H., W.G.T., D.O., D.J.A.), Alfred Hospital, Prahran, Australia; and Department of Medicine, University of Melbourne, St Vincent’s Hospital (D.J.K., R.E.G.), Melbourne, Australia.

Correspondence to Henry Krum, MBBS, PhD, FRACP, NHMRC Centre of Clinical Research Excellence in Therapeutics, Department of Medicine, Monash University Medical School, Alfred Hospital, Commercial Road, Prahran, Victoria, Australia. E-mail henry.krum{at}med.monash.edu.au

	Abstract

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Urotensin II (UII) is a somatostatin-like peptide recently identified as a potent vasoconstrictor. In this study, we examined whether UII promotes cardiac remodeling through nonhemodynamic effects on the myocardium. In a rat model of heart failure after myocardial infarction (MI), increased UII peptide and UII receptor protein expression was observed in both infarct and noninfarct regions of the left ventricle compared with sham. Moreover, post-MI remodeling was associated with a significant 75% increase in UII receptor gene expression in the heart (P<0.05 versus sham controls), with this increase noted in both regions of the left ventricle. In vitro, UII (10^-7 mol/L) stimulation of neonatal cardiac fibroblasts increased the level of mRNA transcripts for procollagens ₁(I), ₁(III), and fibronectin by 139±15% (P<0.01), 59±5% (P<0.05), and 141±14% (P<0.01), respectively, with a concomitant 23±2% increase in collagen peptide synthesis as determined by ³H-proline incorporation (P<0.01). UII had no effect on cellular hypertrophy, as determined by changes in total protein content in isolated neonatal cardiomyocytes. However, expression of recombinant rat UII receptor in neonatal cardiomyocytes resulted in significant UII-dependent activation of hypertrophic signaling as demonstrated by increased total protein content (unstimulated, 122.4±4.0 µg/well; rat UII, 147.6±7.0 µg/well; P<0.01) and activation of the hypertrophic phenotype through Gq- and Ras-dependent pathways. These results indicate that, in addition to potent hemodynamic effects, UII may be implicated in myocardial fibrogenesis through increased collagen synthesis by cardiac fibroblasts and may also be an important determinant of pathological cardiac hypertrophy in conditions characterized by UII receptor upregulation.

Key Words: urotensin • myocardial infarction • remodeling • collagen • hypertrophy

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Left ventricular dysfunction, regardless of the underlying etiology, is associated with activation of various neurohormonal systems that compensate for the consequent loss of ventricular function and peripheral homeostasis. These include the renin-angiotensin and endothelin systems, which act to restore the early decline in cardiac output through positive inotropic effects and peripheral vasoconstriction. Although these effects may be beneficial in the short term, prolonged activation, both local and systemic, may additionally contribute to worsening of contractile function through adverse cardiac remodeling, a process characterized by necrosis,¹ myocyte hypertrophy,² inflammation,³ and the pathological accumulation of extracellular matrix material within the interstitium (fibrosis).⁴

More recently, the vasoconstrictor peptide urotensin II (UII) has emerged as a likely contributor to cardiovascular physiology and pathology. UII is a somatostatin-like cyclic peptide synthesized by proteolytic cleavage from a precursor molecule, prepro-UII, and has recently been identified as a potent vasoconstrictor.⁵ UII has been identified within the heart,⁶ which is also an abundant site of UII receptor (UIIR) expression.⁵ Acute UII infusion into nonhuman primates results in severe myocardial depression, characterized by bradycardia and reduced stroke volume and contractility,⁵ suggesting a functional role for this peptide within the heart. However, the role of UII within the myocardium remains poorly understood, particularly in the setting of disease.

Thus, UII may be an important determinant of cardiac dysfunction via additional nonhemodynamic effects on the myocardium, analogous to those previously described for other vasoconstrictor peptides such as angiotensin II (Ang II) and endothelin 1 (ET-1). Accordingly, this study sought to determine whether UII has direct effects on isolated cardiac cell types in vitro, as well as examining the expression of UII in an experimental model of left ventricular remodeling after myocardial infarction (MI) in the rat. The data demonstrate that UII peptide and receptor expression is upregulated within injured myocardium. Moreover, we show for the first time that UII is profibrogenic in cardiac fibroblast cultures and can regulate hypertrophic signaling in cardiomyocytes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Left Ventricular MI in the Rat
The animal model, as previously described,⁷ conforms to the Guide for the Care and Use of Laboratory Animals published by the NIH (NIH publication No. 85-23, revised 1996). At 1 or 2 weeks after surgery (sham, n=5; left ventricular MI [LVMI], n=5), hemodynamic measurements were performed⁷ and then whole hearts were excised, embedded into OCT embedding compound, and snap frozen over liquid nitrogen for subsequent immunohistochemical analysis and total RNA isolation. Infarct size was determined on cryostat sections as previously described⁷ in detail.

Immunohistochemistry
Immunoreactive UII was assessed using an avidin-biotin technique on either snap-frozen tissue sections postfixed with 4% paraformaldehyde or 10% buffered formalin-fixed, paraffin-embedded tissue sections.⁸ Sections of the left ventricle (LV) were incubated overnight at 4°C with rabbit anti-human UII antibody (a kind gift of Dr S. Douglas, Glaxo SmithKline, King of Prussia, Pa), rat anti-GPR14 antibody (Alpha Diagnostics, San Antonio, Tex), and mouse anti-human mast cell chymase (Chemicon International, Temecula, Calif). Cross reactivity of the UII antibody was absent or minimal against a range of peptide ligands, as previously described.⁹ Negative controls represented either the absence of primary antibody or an irrelevant antibody control.

Neonatal Cardiomyocyte and Fibroblast Cultures
Neonatal cardiomyocytes and fibroblasts, isolated from the hearts of 1-day-old Sprague-Dawley rat pups, were seeded onto 12-well plates at 0.125x10⁴ cells/mm² (high density) in MEM supplemented with 10% FBS and bromodeoxyuridine.¹⁰ After 16 hours, the cells were placed into serum-free defined medium¹⁰ and maintained in this medium thereafter. To prevent spontaneous contraction, cultures were maintained in medium containing 50 mmol/L KCl. Neonatal cardiac fibroblasts were purified by differential plating¹⁰ and used at passage 1 for all experiments.

Measurement of Collagen Synthesis by ³H-Proline Incorporation
In vitro collagen synthesis by neonatal cardiac fibroblasts was determined on confluent cultures by ³H-proline incorporation as previously described.¹¹

Northern Blot Analysis
Levels of procollagen ₁(I) and ₁(III) and fibronectin mRNA transcripts were determined by Northern blot analysis as previously described⁷ using ³²P-labeled cDNA fragments complementary to procollagen ₁(I), ₁(III),⁷ and rat fibronectin generated by reverse transcription–polymerase chain reaction (RT-PCR) from infarcted rat cardiac tissue and spanning positions 1002 to 1578 of the published sequence.¹²

RNase Protection Analysis
Level of UIIR and AT_1A receptor mRNA transcripts was measured by RNase protection analysis as previously described.¹³ cRNA probes were generated from a 449-bp fragment of the rat UIIR cDNA corresponding to nucleotides 799 to 1248¹⁴ and a 618-bp fragment of the rat AT_1A receptor cDNA corresponding to nucleotides -52 to +566. As a control, a 177-nucleotide GAPDH probe was cohybridized in each reaction.

Reverse Transcription–Polymerase Chain Reaction
Rat UIIR gene transcripts were amplified by RT-PCR from DNAse I–treated (0.2 U/µL), reverse-transcribed mRNA from neonatal cardiomyocyte and fibroblasts using sense (5'-GGGCATGGTGGG-AAATGTA-3') and antisense (5'-CGCCGTGTCTGCTTGAAAG-3') primers corresponding to positions 427 and 1014, respectively, of the published sequence.⁵

Quantitative Real-Time PCR
UII gene expression was measured and quantified using the GeneAmp 5700 Sequence Detector (PE Biosystems) according to the manufacturer’s instructions. Primers were obtained from Sigma-Aldrich. The fluorogenic probe (Applied Biosystems) includes a fluorescence reporter (6-carboxyfluorescein [FAM]) at the 5' end and a fluorescent quencher (6-carboxytetramethylrhodamine [TAMRA]) at the 3' end. A commercial, predeveloped 18S control kit labeled with the fluorescent reporter dye [VIC] on the 5' end and the quencher [TAMRA] on the 3' end (PE Biosystems) was used as the endogenous control. The 25-µL PCR mixture contains 12.5 µL of Taqman Univeral PCR Master Mix, 500 nmol/L primers (forward and reverse), 100 nmol/L of Taqman probe, and 1 µL of cDNA template. PCR was performed at 50°C for 2 minutes and 95°C for 10 minutes and then run for 50 cycles at 95°C for 15 seconds and 60°C for 1 minute. Results were expressed as the ratio of UII to 18S relative to sham hearts, which were arbitrarily assigned a value of 1.

Transient Transfection and Reporter Assays
Cardiomyocytes were transfected with 0.45 µg of myosin light chain (MLC)-2v-CAT, atrial natriuretic peptide (ANP) 328-Luc, or skeletal actin (-skACT)-CAT, together with 0.45 µg of the appropriate expression vector for the rat UIIR (rUIIR) (pRc/CMV-rUIIR) using Lipofectamine.¹⁵ All plates received equivalent amounts of DNA (0.9 µg) by cotransfection, where necessary, with the appropriate empty expression vector. Twenty-four hours after transfection, cells were stimulated with vehicle, UII, or phenylephrine (PE) (for 72 hours) and harvested for reporter assays.¹⁵

Generation of Recombinant Adenovirus
Recombinant adenovirus directing the expression of the rat UIIR (Ad-rUIIR) was generated by bacterial homologous recombination, as described.^16,17 Twenty-four hours after plating, cells were infected with purified virus at a multiplicity of infection (MOI) of 12.5 to 300 in defined medium as indicated.¹⁷ An MOI of 100 corresponds to 1x10⁸ PFU of virus, which infected greater than 95% of 1x10⁶ myocytes and fibroblasts as defined by green fluorescent protein fluorescence. After 24 hours of infection, the medium was replaced and cells were assayed in accordance with the previously outlined methodologies.

Urotensin II Receptor Binding Assay
Expression of UII receptors in cultures of neonatal cardiomyocytes and fibroblasts after infection with adenovirus (Ad-rUIIR) was determined using a whole-cell binding assay¹⁸ with radiolabeled ([¹²⁵I])-rat UII. [¹²⁵I]-rat UII was prepared (specific activity 1000 Ci/mmol, Austin Biomedical Services) from HPLC purified rat UII peptide (kindly synthesized, cyclized, purified, and authenticated using mass spectrometry by K. Stewart, Monash University and C. Hamilton, Baker Medical Research Institute).

Statistical Analysis
Nonparametric analysis by Mann-Whitney U test was applied to all data sets, with a P<0.05 representing statistical significance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Myocardial Expression of UII and UIIR After MI
MI rats had moderate to large infarcts (34.7±0.5% of length of LV epicardial and endocardial circumferences). In comparison with sham, MI animals demonstrated characteristic hemodynamic changes, with a markedly elevated LV end diastolic pressure (19.4±0.03 versus 9.2±0.02 mm Hg, P<0.05), reduced LV dP/dt_max (5.4±0.8 versus 8.3±0.7 mm Hg/ms, P<0.05), and reduced mean arterial pressure (86±4 versus 111±5 mm Hg, P<0.05).

UII Receptor
RNase protection analysis demonstrated a significant 80±11% increase in UIIR mRNA transcripts in whole heart by 1 week after MI compared with sham controls (Figure 1A). In comparison, AT_1A receptor mRNA was increased by 175±35%. Additional analysis by RT-PCR showed that both neonatal cardiomyocyte and nonmyocyte populations (predominantly fibroblasts) constitutively express rat UIIR mRNA transcripts (Figure 1B).

View larger version (32K): [in this window] [in a new window]   Figure 1. A, rUIIR mRNA transcripts are increased in the rat heart at 1 week after MI (n=6). Total RNA 25 µg isolated from whole rat heart was assayed for rUIIR mRNA transcripts by RNase protection analysis. Comparison was also made to angiotensin II receptor AT1 mRNA. RNase protected bands were visualized and quantitated by phosphorimaging, with results represented as a ratio to GAPDH (mean±SEM). *P<0.05 vs sham controls. B, rUIIR gene expression in isolated neonatal cardiomyocytes (MC) and nonmyocytes (NMC; predominantly fibroblasts) by RT-PCR. Amplified PCR products were visualized by FluorImaging after agarose gel electrophoresis. M=/HindIII molecular markers. C, RNase protection assay for UIIR. Data for each heart zone (n=3) are expressed as a fold change above the corresponding zone from the sham. Lane 1, Sham/right ventricle; lane 2, sham/noninfarct zone equivalent of LV; lane 3, sham/infarct zone equivalent of LV; lane 4, right ventricle in MI animals at 1 week; lane 5, MI/noninfarct zone of LV; and lane 6, MI/infarct zone of LV. Top two panels are RPA quantification, and insets are the phosphorimages for UII and ANP as positive control. Bottom panel is total RNA showing 28S and 18S bands.

Assessment of the distribution of this increase in UIIR expression was performed by RNase protection analysis. This demonstrated increased expression of UIIR in both the infarct and noninfarct zones of the LV as well as in right ventricle (Figure 1C). ANP expression was also measured as a marker gene known to be activated in the post-MI setting (Figure 1C).

Immunohistochemistry using anti-GR14 antibody demonstrated that UIIR was not detectable in the myocardium of sham rats (Figure 2A) whereas post-MI UIIR was localized to myocytes (Figure 2B), endothelial cells, and fibroblasts (Figure 2C).

View larger version (84K): [in this window] [in a new window]   Figure 2. Immunohistochemistry using anti-GR14 antibody (magnification x380) demonstrated that UIIR was not detectable in the myocardium of sham rats (A), whereas after MI, UIIR was localized to myocytes (B), endothelial cells (C), and fibroblasts (C, arrow).

UII Peptide
In normal and sham-operated rat heart, UII peptide was barely detected and immunolocalized predominantly to the vasculature (Figure 3a). In contrast, animals with MI demonstrated increased cellular expression of UII peptide within the left ventricular infarct region (Figures 3c and 3d) and increased diffuse staining within the interstitium of peri-infarct regions (Figures 3e and 3f). Additional analysis revealed that cellular UII expression within the infarct region was localized to chymase-positive mast cells (Figures 3g and 3h). Pre-pro UII peptide gene expression as determined by quantitative real-time PCR revealed increased UII mRNA in both infarct and noninfarct zones of the LV compared with sham animals (Ln-transformed fold increases compared with sham: right ventricle, 4.29; left ventricular noninfarct zone, 5.98; left ventricular infarct zone, 4.15). No significant correlations were observed between UII or UIIR expression and hemodynamic parameters or infarct size.

View larger version (82K): [in this window] [in a new window]   Figure 3. Immunoreactive UII peptide is increased within infarct and noninfarct regions at 2 weeks after MI. a, Snap-frozen, paraformaldehyde-fixed section of normal rat heart demonstrating UII staining within vessels. In contrast to the absence of staining in the negative control (b), animals with MI display increased UII peptide expression within discrete cells of the infarcted myocardium (c through d). Boxed area of c is shown with higher magnification in d. e, Section of normal rat heart demonstrating little UII peptide expression within the myocardial interstitium. f, MI animals showing increased diffuse staining for UII within peri-infarct regions of the myocardium (scale bar=100 µm). Immunoreactive UII is colocalized to chymase-positive mast cells within the infarcted myocardium. Consecutively sliced, paraffin-embedded tissue sections of rat heart harvested from 2-week post-MI animals stained for mast cell chymase (g) and rat UII (h). Scale bar=100 µm.

Effect of UII on Collagen Synthesis by Neonatal Cardiac Fibroblasts In Vitro
Compared with medium alone, UII stimulated an increase in fibronectin, ₁(I), and ₁(III) procollagen mRNA transcripts in neonatal cardiac fibroblasts (Figure 4A). This was associated with a concomitant increase in collagen synthesis, whereas there was no discernible synergistic effect with Ang II (Figure 4B).

View larger version (28K): [in this window] [in a new window]   Figure 4. UII stimulates matrix synthesis by neonatal rat cardiac fibroblasts in vitro. A, Fibronectin and procollagen (I) and (III) mRNA were measured by Northern blot analysis after 24-hour stimulation with ET-1 (10-7 mol/L) and rUII (10-7 mol/L). Data represent samples analyzed over 4 separate experiments, and the results are expressed as a mean percentage of unstimulated controls (C) (±SEM) relative to GAPDH (n=12). B, Collagen peptide synthesis was determined by standard 3H-L-proline incorporation after 48-hour stimulation with Ang II (10-7 mol/L), rUII (10-7 mol/L), or combined stimulation in DMEM-F12 medium supplemented with 0.15 mmol/L ascorbic acid. Data represent samples analyzed over 3 separate experiments, and the results are expressed as a mean percentage of unstimulated controls (C) (±SEM) (n=9). *P<0.05, **P<0.01 vs unstimulated controls.

Introduction of recombinant rUIIR introduced into cells using adenoviral vectors resulted in a dose-dependent increase in [¹²⁵I]-UII binding compared with noninfected cultures (Figure 5A). Moreover, cells infected with even the lowest dose of Ad-rUIIR (MOI 12.5) demonstrated an even greater profibrogenic response to exogenous UII (Figure 5B) than noninfected cells. These results clearly demonstrate that modest overexpression of the UIIR enhances the profibrogenic stimulus of UII in neonatal cardiac fibroblasts. UII lacked a mitogenic effect in primary neonatal cardiac fibroblasts or in cells overexpressing recombinant rUIIR (data not shown).

View larger version (22K): [in this window] [in a new window]   Figure 5. Adenoviral-mediated increase in rUIIR expression enhances the profibrogenic response in neonatal cardiac fibroblasts in vitro. A, Cells infected with Ad-rUIIR demonstrate a dose-dependent increase in whole-cell binding of radiolabeled [125I]-rUII. Results are expressed as mean (±SEM) counts per minute (cpm) minus nonspecific cpm determined by the addition of excess unlabeled rUII (10-6 mol/L) (n=6). B, Collagen peptide synthesis was determined by 3H-L-proline incorporation after 48-hour stimulation with rUII (10-7 mol/L) in either primary cultures or those preinfected for 24 hours with Ad-rUIIR at an MOI of 12.5. The results are expressed as a mean percentage of unstimulated controls (C) (±SEM) (n=6). **P<0.01 vs unstimulated controls.

Effect of UII on Cardiac Myocyte Hypertrophy In Vitro
In contrast to the hypertrophic stimulus of ET-1 and PE (total protein content [µg/well]; unstimulated, 10.2±3.0; ET-1, 16.0±1.9; PE, 13.7±0.4; P<0.01), rUII had no effect on myocyte hypertrophy in high-density cultures (rUII [10^-7 mol/L], 10.5±0.8 µg). In each case, the inability of UII to stimulate hypertrophy was not attributable to loss of cell number, because DNA content was unchanged (unstimulated, 2.98±0.1; ET-1, 3.11±0.3; PE, 3.05±0.2; rUII, 3.12±0.2).

In support of the hypothesis that levels of rUIIR in primary neonatal cardiomyocytes may be below the threshold required for a hypertrophic response, we found that in cells transiently transfected with recombinant rUIIR, UII stimulated the activity of several cotransfected reporters of the hypertrophic phenotype (atrial natriuretic C peptide,¹⁹ ventricular MLC-2,²⁰ -skACT²¹) (Figure 6). Furthermore, cotransfection of a construct expressing either the N17-Ras²² or GqI²³ with the ANP promoter reporter and recombinant rUIIR resulted in a significant, though not complete, inhibition of PE- and UII-mediated activation of ANP expression (Figure 7).

View larger version (22K): [in this window] [in a new window]   Figure 6. UII stimulates a hypertrophic phenotype in neonatal rat cardiomyocytes transiently transfected with recombinant rUIIR. Stimulation of hypertrophic signaling in cardiomyocytes was measured by activation of promoters for ANP, MLC-2, and -skACT (percent increase in luciferase [ANP, MLC-2]/chloramphenicol transferase [skACT] activity) after 24-hour stimulation with either PE (10-4 mol/L) or rUII (10-7 mol/L), n=5. Results are expressed as a mean percentage increase in enzyme activity over unstimulated controls (C) (±SEM). Transfection combinations are outlined along the x-axis. *P<0.001 vs relevant control.

View larger version (20K): [in this window] [in a new window]   Figure 7. UII-mediated activation of the ANP promoter in neonatal cardiomyocytes transfected with rUIIR is partly mediated via Gq and Ras. Stimulation of hypertrophic signaling (ANP promoter activity) in isolated cardiomyocytes cotransfected with either vector alone (pRc/CMV and pRK5) or combinations of rUIIR, N17Ras, or GqI constructs was measured after 24-hour stimulation with PE (10-4 mol/L) or rUII (10-7 mol/L), n=5. Results are expressed as a mean percentage increase in luciferase activity over unstimulated controls (C) (±SEM). Transfection combinations are outlined along the x-axis.

To document increased cell protein content as a marker of hypertrophic growth, we introduced recombinant rUIIR cDNA into neonatal cardiomyocytes using adenoviral vectors, thus introducing recombinant rUIIR into greater than 95% of cultured cells compared with Lipofectamine (typically 5%). Infection of cardiomyocytes with Ad-rUIIR at an MOI of as little as 12.5 resulted in a modest, yet significant, increase in [¹²⁵I]-UII binding in comparison to noninfected cultures. Moreover, the Ad-rUIIR–infected cells demonstrated a marked hypertrophic growth response to UII (total protein content [µg/well]; unstimulated, 122.4±4.0; rUII, 147.6±7.0; P<0.01) equivalent to that observed after stimulation with PE (155.5±6) (Figure 8) with little change in DNA content (µg/well; unstimulated, 3.21±0.24; PE, 3.31±0.3; rUII, 3.16±0.1).

View larger version (18K): [in this window] [in a new window]   Figure 8. UII stimulates hypertrophic growth in neonatal cardiomyocytes infected with recombinant Ad-rUIIR. Cardiomyocytes infected with Ad-rUIIR (MOI 12.5), but not uninfected cells, demonstrate hypertrophic growth in response to rUII (10-7 mol/L) as determined by increased mean cell protein content (±SEM), n=5. PE stimulation (10-4 mol/L) served as a positive control for hypertrophic growth; unstimulated cells served as negative controls. *P<0.01 vs negative control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Myocardial injury is associated with significant structural remodeling, including cardiac hypertrophy and marked interstitial fibrosis.²⁴ Vasoconstrictor peptides such as Ang II and ET-1 have emerged as likely candidates²⁵ in these processes. Thus, we sought to determine whether similar actions could be described for the newly identified vasoconstrictor peptide UII.

The present study identified the presence of UII and its receptor within myocardial tissue and suggests an important role for UII in cardiac pathophysiology. This is supported by our findings that both UII and its receptor are upregulated after MI in the rat, with these increases distributed to both the infarct and noninfarct zone of the LV. Although this increase in UIIR was observed in all regions measured, it was highest in the right ventricle, consistent with increased pulmonary pressures secondary to LVMI or loss of the myocyte population of cells within the LV, resulting in reduced LV gene expression in this area.

UII peptide was seen in a subpopulation of mast cells. It is of interest that mast cells, identified in larger numbers in both human²⁶ and rat heart²⁷ after ischemic injury, seem to play an important role in the evolution of decompensated cardiac hypertrophy and perivascular fibrosis.²⁸

The above observations are consistent with those of Douglas et al,⁹ who have reported increased expression of UII and its receptor in the myocardium of patients with congestive heart failure. The findings of the present study, with increases in UII and UIIR observed soon after myocardial injury, suggest a direct pathophysiological role for the UII system in cardiac remodeling.

An increase in UIIR peptide was observed throughout the myocardium and localized to myocytes, endothelial cells, and fibroblasts, supporting a functional role for UII in cardiac remodeling, acting via these cell types. To explore this potential role for the UII system additionally, we examined the effect of exogenous UII on isolated cardiac cell types in vitro. Analogous to the profibrogenic effects previously documented for other vasoconstrictor peptides, such as Ang II and ET-1,²⁵ we have found that exogenous UII also stimulates increased matrix synthesis by neonatal cardiac fibroblasts, a key cell type implicated in pathological matrix synthesis within the myocardium.²⁹ Moreover, this effect was enhanced in the setting of modest UIIR overexpression, suggesting that UII-mediated profibrogenic responses in the post-MI setting could be regulated through altered UIIR expression by resident cardiac fibroblasts and myofibroblasts.

Vasoconstrictor peptides have also been shown to regulate cardiac hypertrophy in response to increased workload, an effect that is mediated predominantly through hypertrophic growth of cardiomyocytes.³⁰ However, we found that UII does not exhibit an analogous hypertrophic stimulus in quiescent myocytes cultures, as measured by changes in total protein content or in the activation of reporters of the hypertrophic phenotype.

Additional study suggests that the level of rUIIR expression in neonatal cardiomyocytes is below the threshold required for the sustained signaling required for the hypertrophic response. Indeed, in our hands using cultured neonatal cardiomyocytes, even the established hypertrophic hormone Ang II fails to promote hypertrophy¹⁸; however, infection with an adenovirus encoding the AT₁ receptor was necessary to produce a reproducible hypertrophic response to Ang II, strongly implicating receptor upregulation as the defining determinant for initiating hypertrophy.¹⁸ In support of the hypothesis, we found that in cardiomyocytes expressing modest levels of recombinant rUIIR, UII stimulated hypertrophic growth as determined by increased cell protein content as well as activation of phenotypic markers of the hypertrophic response. This effect was mediated, in part, through Gq- and Ras-dependent pathways, analogous to the hypertrophic signaling pathways of other 7-transmembrane-spanning G protein–coupled receptors.¹⁸

Although these observations are made in the in vitro setting (with obvious caution required in their translation into the in vivo setting), these results strongly suggest that, at least in the rat model, the UII system is capable of stimulating cardiac hypertrophy under conditions of receptor upregulation. The potential pathophysiological relevance of this observation is supported by our findings that UIIR gene expression is significantly increased after MI. Similar observations have been made in established human heart failure.⁹ It is also tempting to speculate that potential upregulation of the UIIR under certain other pathological conditions, such as pressure overload, may contribute to the resultant hypertrophic response. Indeed, upregulation of receptors for other vasoconstrictor peptides has been suggested as a mechanism by which hypertrophic growth is mediated after cardiac injury.³¹

In conclusion, this study describes, for the first time, upregulation of UII and UIIR in the post-MI setting and profibrogenic effects of UII in isolated cardiac fibroblasts. The present study also suggests that UII may be an important determinant of cardiac hypertrophy in conditions characterized by UIIR upregulation. Moreover, these studies reveal that the intracellular signaling pathways linked to the profibrogenic and hypertrophic effects are likely to be mediated via Gq- and Ras-dependent pathways. Although future studies are required to assess the contribution of endogenous UII on adverse cardiac remodeling in vivo (particularly with the use of specific UIIR antagonists), this report highlights the analogies between UII and other vasoconstrictor peptides implicated in this process, thus supporting a role for UII in the pathogenesis and progression of cardiovascular disease.

	Acknowledgments

 
This study was supported by National Health and Medical Research Council of Australia Project Grant Nos. 194429 and 166900. The authors would like to thank A. Jenkins for her technical assistance.

	Footnotes

 
Original received February 20, 2003; revision received June 19, 2003; accepted June 19, 2003.

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