Am J Physiol Heart Circ Physiol 301: H2372–H2382, 2011.

First published September 16, 2011; doi:10.1152/ajpheart.00283.2011.

A role for cardiotrophin-1 in myocardial remodeling induced by aldosterone

Natalia López-Andrés,1 Beatriz Martin-Fernandez,2 Patrick Rossignol,1,3 Faiez Zannad,1,3 Vicente Lahera,2
Maria Antonia Fortuno,4 Victoria Cachofeiro,2 and Javier Díez4,5
1
U961, Faculty of Medicine, Institut National de la Santé et de la Recherche Médicale (INSERM), Vandoeuvre-lès-Nancy,
France; 2Departamento de Fisiologia. Universidad Complutense de Madrid, Madrid, Spain; 3Clinical Investigation Centre,
CIC9501, INSERM, Vandoeuvre-lès-Nancy, France; and 4Division of Cardiovascular Sciences, Centre for Applied Medical
Research, and 5Department of Cardiology and Cardiac Surgery, University Clinic, University of Navarra, Pamplona, Spain
Submitted 23 March 2011; accepted in final form 18 August 2011

López-Andrés N, Martin-Fernandez B, Rossignol P, Zannad F,
Lahera V, Fortuno MA, Cachofeiro V, Díez J. A role for cardiotro-
phin-1 in myocardial remodeling induced by aldosterone. Am J
Physiol Heart Circ Physiol 301: H2372–H2382, 2011. First published
September 16, 2011; doi:10.1152/ajpheart.00283.2011.—Hyperaldo-
steronim is associated with left ventricular (LV) hypertrophy (LVH)
and fibrosis. Cardiotrophin (CT)-1 is a cytokine that induces myocar-
dial remodeling. We investigated whether CT-1 mediates aldosterone
(Aldo)-induced myocardial remodeling in two experimental models.
Wistar rats were treated with Aldo-salt (1 mg·kg⫺1·day⫺1) with or
without spironolactone (200 mg·kg⫺1·day⫺1) for 3 wk. Wild-type
(WT) and CT-1-null mice were infused with Aldo (1 mg·kg⫺1·day⫺1)
for 3 wk. Hemodynamic parameters were analyzed. LVH, fibrosis,
inflammation, and CT-1 expression were evaluated in both experi-
mental models by histopathological analysis, RT-PCR, Western blot
analysis, and ELISA. Hypertensive Aldo-treated rats exhibited in-
creased LV end-diastolic pressure and ⫺dP/dt compared with con-
trols. The cardiac index, LV cross-sectional area and wall thickness,
cardiomyocyte size, collagen deposition, and inflammation were in-
creased in Aldo-salt-treated rats. Myocardial expression of molecular
markers assessing LVH and fibrosis as well as CT-l levels were also
augmented by Aldo-salt. Spironolactone treatment reversed all the
above effects. CT-1 correlated positively with hemodynamic, histo-
logical, and molecular parameters showing myocardial remodeling. In
WT and CT-1-null mice, Aldo infusion did not modify blood pressure.
Whereas Aldo treatment induced LVH, fibrosis, and inflammation in
WT mice, the mineralocorticoid did not provoke cardiac remodeling
in CT-1-null mice. In conclusion, in experimental hyperaldosteron-
ism, the increase in CT-1 expression was associated with parameters
showing LVH and fibrosis. CT-1-null mice were resistant to Aldo-
induced LVH and fibrosis. These data suggest a key role for CT-1 in
cardiac remodeling induced by Aldo independent of changes in blood
pressure levels.
fibrosis; left ventricular hypertrophy; inflammation
ALDOSTERONE (Aldo) is a mineralocorticoid hormone that has
been associated with the development of cardiovascular re-
modeling, fibrosis, and injury (1, 2, 4, 27, 35). Myocardial
fibrosis and left ventricular (LV) hypertrophy (LVH) are hall-
marks of most cardiac pathologies and facilitate the develop-
ment of heart failure (HF) (25, 29). Accumulating clinical
evidence shows that an excess of Aldo in patients with primary
hyperaldosteronism is associated with LVH and alterations in
myocardial texture (23, 24). Moreover, blockade of the min-
eralocorticoid receptor has been shown to improve outcome,
LVH, and fibrosis in patients with HF (22, 36, 37).
Address for reprint requests and other correspondence: N. López-Andrés,
INSERM, U961, Faculty of Medicine, Vandoeuvre-lès-Nancy 54505, France
(e-mail: natalia.lopez-andres@nancy.inserm.fr).
Cardiotrophin (CT)-1 is a member of the IL-6 superfamily
that exerts its cellular effects by interacting with the het-
erodimer constituted by the glycoprotein 130 and leukemia
inhibitory factor receptor-␤ (21). Our group (13, 14) has
already characterized CT-1-induced cardiomyocyte survival
and hypertrophy. Moreover, it has been shown that CT-1
directly stimulates cardiac fibroblast proliferation and collagen
type I synthesis (5, 31), suggesting a role for the cytokine in the
development of myocardial fibrosis.
Our group (9, 10) has recently shown that Aldo induced
CT-1 expression in both cardiac and vascular cells in vitro.
Moreover, Aldo-induced CT-1 upregulation seems to play a
role in the ability of the mineralocorticoid to produce cardio-
myocyte growth and, as a result, LVH (10). Furthermore,
elevated plasma concentrations of CT-1 have been reported in
HF patients (12), and a significant association has been found
between abnormally high CT-1 and abnormally high Aldo in
these patients (12), suggesting that the mineralocorticoid path-
ways might be involved in CT-1 overproduction in HF.
Therefore, the hypothesis emerges that CT-1 could be a key
factor involved in the cardiovascular remodeling induced by
Aldo associated with cardiac hypertrophy and fibrosis, facili-
tating cardiovascular dysfunction. The present study was de-
signed to examine the role of CT-1 in myocardial remodeling
induced by Aldo in two animal models: 1) rats treated with
Aldo-salt and 2) wild-type (WT) and CT-1-null mice infused
with Aldo under a normal Na⫹ diet.
METHODS
Animals. This investigation was carried out with governmental
approval (license no. B 54-547-20) and was performed in accordance
with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals (NIH Pub. No. 82-23, Revised 1996). Twelve-
week-old male Wistar rats weighing 250 g (Harlan Ibérica, Barcelona,
Spain) were randomly divided into four groups. In the control group
(n ⫽ 13), rats received subcutaneous vehicle (sunflower oil) for 3 wk.
In the Aldo-salt group (n ⫽ 17), rats received subcutaneous Aldo
dissolved in sunflower oil (1 mg·kg⫺1·day⫺1) and 1% NaCl as
drinking water for 3 wk. In the Aldo-salt ⫹ spironolactone (Spiro)
group (n ⫽ 8), rats received subcutaneous Aldo (1 mg·kg⫺1·day⫺1)
and Spiro (200 mg·kg⫺1·day⫺1) and 1% NaCl as drinking water. Finally, in
the Spiro group (n ⫽ 5), rats received subcutaneous Spiro (200
mg·kg⫺1·day⫺1). The model and dosage of Aldo were chosen from
pilot studies in which the treatment increased blood pressure and
induced LVH. The plasma Aldo concentration was analyzed by a
specific quantitative sandwich enzyme immunoassay (Cayman Chem-
ical, Cayman, MI).
Adult CT-1-null mice backcrossed into a C57BL6 background were
obtained from Dr. Pennica (Genentech). The experimental groups were
always composed of littermate animals. Aldo-treated WT and CT-1-null
mice were implanted with osmotic mini-pumps (model 2004, Alzet, 1

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 CARDIOTROPHIN-1 MEDIATES ALDOSTERONE-INDUCED CARDIAC REMODELING H2373
Table 1. Effects of Aldo in general and hemodynamic RT and real-time PCR. Total RNA extraction was performed using
parameters in rats a nucleic acid purification lysis solution (Applied Biosystems) and the
semiautomated ABI Prism 6100 Nucleic Acid PrepStation system
Parameter Control Aldo Aldo ⫹ Spiro (Applied Biosystems). Real-time PCR was performed with an ABI
n 13 17 8
PRISM 7000 Sequence Detection System using specific TaqMan
BW, g 341 ⫾ 10 334 ⫾ 5 322 ⫾ 4 MGB fluorescent probes (Applied BioSystems). Constitutive 18S
HW, g 1.12 ⫾ 0.02 1.23 ⫾ 0.02 * 1.03 ⫾ 0.03 rRNA was used as an endogenous control.
HW/BW, mg/g 3.3 ⫾ 0.09 3.7 ⫾ 0.12 * 3.2 ⫾ 0.11 Western blot analysis. LVs were homogenized in lysis buffer
SBP, mmHg 118 ⫾ 2 141 ⫾ 4 * 122 ⫾ 2 (Roche). Protein concentrations were determined by the Bradford
DBP, mmHg 88 ⫾ 3 100 ⫾ 4 * 90 ⫾ 3 method (Bio-Rad). Proteins (30 ␮g) were separated by SDS-PAGE
MAP, mmHg 97 ⫾ 2 113 ⫾ 4 * 100 ⫾ 3 and transferred to nitrocellulose membranes (Amersham Biosciences).
LVEDP, mmHg 3.6 ⫾ 0.3 9.3 ⫾ 0.6 * 3.6 ⫾ 0.3 Blots were visualized using the ECL-Plus chemiluminescence detec-
LVSP, mmHg 122 ⫾ 3 140 ⫾ 5 * 114 ⫾ 2 tion system (Amersham Biosciences).
⫹dP/dt, mmHg/s 9,379 ⫾ 358 8,932 ⫾ 236 8,128 ⫾ 388
⫺dP/dt, mmHg/s ⫺6,455 ⫾ 173 ⫺5,324 ⫾ 218 * ⫺6,000 ⫾ 230
Immunohostochemistry. Myocardial sections (5 ␮m thick) were de-
HR, beats/min 294 ⫾ 22 233 ⫾ 12 * 254 ⫾ 10 waxed and rehydrated, and slides were heated for 10 min in a solution
containing 10 mM sodium citrate (pH 6.0) for CT-1 immunodetection or
Values are means ⫾ SE; n, no. of animals. Aldo, aldosterone; Spiro, with 10 mM Tris-EDTA (pH 9.0) for CD3 immunodetection, incubated
spironolactone; BW, body weight; HW, heart weight; SBP, systolic blood in 1% H2O2 for 10 min, and blocked with 5% normal goat serum in PBS
pressure; DBP, diastolic blood pressure; MAP, mean arterial pressure; for 1 h. Slides were incubated overnight with CT-1 antibody (1:20
LVEDP, left ventricular (LV) end-diastolic pressure; LVSP, LV systolic
pressure; ⫹dP/dt, first derivative of the LV pressure rise over time; ⫺dP/dt,
dilution, Abcam) or CD3 antibody (1:50 dilution, Dako Cytomation),
first derivative of the LV pressure decline over time; HR, heart rate. *P ⬍ 0.01 washed three times, and then incubated for 30 min with the horseradish
vs. control. peroxidase-labeled polymer conjugated to secondary antibodies (Dako
Cytomation). The signal was revealed using diaminobenzidine chroma-
gen substrate (Dako Cytomation), and slides were counterstained with
mg·kg⫺1·day⫺1) for subcutaneous infusion for 3 wk. Control animals hematoxylin (Sigma-Aldrich). Myocardial sections from CT-1-null mice
received PBS instead of Aldo. All animals received standard chow were used as negative controls.
without the addition of excess dietary salt to avoid the hemodynamic ELISA. Plasma levels of CRP and TNF-␣ in rats and mice and
effects. myocardial levels of CT-1 in mice were determining using commer-
Hemodynamic parameters. Anesthesia was induced with ketamine cially available kits (R&D Systems). All samples were run in dupli-
(70 mg/kg ip) and xylazine (Rompun, 6 mg/kg ip), and a catheter cate with the average of the two replicates reported.
(Science FT211B, 1.5-mm diameter) was advanced into the right Statistical analysis. Results are presented as means ⫾ SE. Compar-
ventricle through the right carotid artery. The catheter was connected isons between groups of animals were made by one-way ANOVA
to a data-acquisition system (PowerLab/800, AD Instruments), and followed by a Scheffé test. P values lower than 0.05 were considered
signals were monitorized and digitally stored for analysis with Chart significant. The relation between variables was tested calculating the
for Windows software. The following parameters were evaluated: Pearson correlation coefficient and, when applicable, the Spearman cor-
systolic blood pressure (SBP), diastolic blood pressure (DBP), LV relation coefficient. Multivariable linear regression models were used to
end-diastolic pressure (LVEDP), LV systolic pressure (LVSP), the assess the independent relation between CT-1 and parameters of LVH
first derivative of the LV pressure rise over time (⫹dP/dt), and the first and fibrosis after an adjustment for blood pressure, which has previously
derivative of the LV pressure decline over time (⫺dP/dt). been found to be associated with LVH fibrosis in univariable regression
Telemetry. The surgical procedure for transmitter implantation was models. Differences in responses to Aldo between WT and CT-1-null
performed in a sterilized area. Anesthesia was induced with 3% mice were assessed by a two-factor ANOVA test. Comparisons were
isofluorane-O2 and maintained during surgery with 1.5%. Telemetry adjusted for multiple testing using Scheffé’s adjustment.
transmitters (model TA11PA-C40, Data Sciences) were implanted
according to the manufacturer’s instructions (Data Sciences), with RESULTS
RCP-1 receivers used for telemetric acquisition and analysis of SBP,
DBP, mean arterial pressure (MAP), and heart rate. The system was Effects of Aldo-salt treatment in general and hemodynamic
scheduled to obtain and store a 10-s measurement every 15 min. Data parameters in rats. The efficiency of the Aldo-salt perfusion
were averaged in 24-h intervals for post hoc analysis. was verified by the increase of blood pressure in Aldo-salt-
Morphological and histological evaluation. Hearts were arrested in
diastole using KCl before being harvested and then fixed and included
in paraffin. Histological determinations in cardiac tissue were per- Table 2. Effects of Aldo in histological and molecular
formed in 5-␮m-thick sections. In Masson’s trichrome-stained sec- parameters assessing myocardial remodeling in rats
tions, the LV cross-sectional area (LVCSA) and LV wall thickness
(LVWT) were measured. For cell width and length, at least 30 Parameter Control Aldo Aldo ⫹ Spiro
cardiomyocytes/section were measured in triplicate. Cardiomyocyte
n 13 16 7
length was measured only in cardiomyocytes where intercalated disks
LVCSA, mm2 37.9 ⫾ 1.2 43.9 ⫾ 1.5* 37.7 ⫾ 1.1
were visible. All measurements were performed blind using an auto- LVWT, mm 2.8 ⫾ 0.08 3.2 ⫾ 0.09* 2.5 ⫾ 0.10
mated image-analysis system (Quancoul). Images were calibrated Cardiomyocyte length, ␮m 97.1 ⫾ 1.6 111.8 ⫾ 2.3* 99.1 ⫾ 2.8
with known standards. Cardiomyocyte width, ␮m 31.5 ⫾ 0.8 40.2 ⫾ 1.4* 32.9 ⫾ 0.4
Sirius red-stained sections were analyzed under an automatized mi- Atrial natriuretic peptide
croscope (⫻40), and all fields covering the myocardial tissue section mRNA, AU 1.3 ⫾ 0.2 3.7 ⫾ 0.5* 1.3 ⫾ 0.2
were digitized. The area of interstitial fibrosis was identified after exclud- c-fos mRNA, AU 3.9 ⫾ 1 42.9 ⫾ 13* 29.4 ⫾ 8†
ing the vessel area from the region of interest, as the ratio of interstitial c-Fos protein, AU 2.4 ⫾ 0.8 9.4 ⫾ 2.6* 3.6 ⫾ 0.4
fibrosis or collagen deposition to the total tissue area. Perivascular fibrosis c-Myc protein, AU 1.8 ⫾ 0.6 9.9 ⫾ 2.9* 2.3 ⫾ 0.6
was analysed in Masson’s trichrome-stained sections. Values are means ⫾ SE; n, no. of animals. LVCSA, LV cross-sectional area;
In Masson’s trichrome-stained sections, the number and area of LVWT, LV wall thickness; AU, arbitrary units. *P ⬍ 0.01 vs. control; †P ⬍
focal inflammatory lesions per section were obtained. 0.05 vs, control.

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H2374 CARDIOTROPHIN-1 MEDIATES ALDOSTERONE-INDUCED CARDIAC REMODELING

treated rats compared with control rats. Moreover, at death,
plasma Aldo was significantly increased in Aldo-salt-treated
animals compared with control animals (700 ⫾ 144 vs. 275 ⫾
42 pg/ml, P ⬍ 0.01).
Average body weight (BW) and heart weight (HW) as
well as blood pressure and cardiac function for control, Aldo-
salt-, and Aldo-salt ⫹ Spiro-infused rats are shown in Table 1.
HW and the HW-to-BW ratio (HW/BW) of Aldo-treated rats
were significantly increased (P ⬍ 0.01) relative to control rats.
Moreover, Aldo-salt induced an increase (P ⬍ 0.01) in SBP,
DBP, and MAP. Values of LVSP were higher (P ⬍ 0.01) in the
Aldo-salt-treated group and ⫹dP/dt was similar in both groups,
indicating a preserved systolic function after Aldo-salt treat-
ment. However, LVEDP and ⫺dP/dt were augmented (P ⬍
0.01) in rats treated with Aldo-salt, demonstrating an altered
diastolic function. Moreover, heart rate was decreased in Aldo-
salt-treated rats compared with control rats. Cardiac hypertro-
phy and hypertension as well as diastolic dysfunction were
fully prevented by Spiro treatment. All the above parameters
were unaffected by Spiro alone (data not shown).
Effects of Aldo-salt treatment in myocardial remodeling in
rats. Cardiac histological and molecular analyses are shown in
Table 2. Aldo-salt-treated rats exhibited increased LVCSA
(16%, P ⬍ 0.01) and LVWT (14%, P ⬍ 0.01) compared with

Fig. 1. Morphology and composition of the myocardium from control, aldosterone (Aldo)-salt, and Aldo-salt ⫹ spironolactone (Spiro)-treated rats. A: representative
photomicrographs of myocardial Masson’s trichrome-stained sections. B: interstital myocardial fibrosis was evaluated in Sirius red-stained sections. After 3 wk of
treatment, interstitial collagen deposition was increased in Aldo-salt-treated animals. Spiro blocked Aldo-salt-induced interstitial fibrosis. C: perivascular myocardial
fibrosis was quantified in sections stained with Sirius red. Aldo-salt-treated rats presented augmented perivascular fibrosis compared with control and Aldo-salt ⫹ Spiro
rats. D: the expression of extracellular matrix components was quantified by RT-PCR and Western blot analysis. 18S rRNA gene expression or ␤-actin levels were used
as loading controls in RT-PCR and Western blot analysis, respectively. Aldo-salt-treated rats presented increased ␣1-procollagen, collagen type I-to-III ratio, and matrix
metalloproteinase (MMP)-13-to-tissue inhibitor of metalloproteinase (TIMP)-1 ratio. *P ⬍ 0.01 vs. control.

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 CARDIOTROPHIN-1 MEDIATES ALDOSTERONE-INDUCED CARDIAC REMODELING
control rats (representative photographs of heart sections are
shown in Fig. 1A). At the cellular level, Aldo-salt but not
vehicle treatment increased cardiomyocyte length (15%, P ⬍
0.01) and cardiomyocyte width (28%, P ⬍ 0.01). Finally, at the
molecular level, Aldo-salt treatment enhanced mRNA expres-
sion of atrial natriuretic peptide (2.8-fold, P ⬍ 0.01) and c-fos
(11-fold, P ⬍ 0.01) and protein expression of c-Fos (4-fold,
P ⬍ 0.01) and c-Myc (5.5-fold, P ⬍ 0.01). Spiro treatment
completely prevented myocardial hypertrophy at both the his-
tological and molecular levels. All the above parameters were
unaffected by Spiro alone (data not shown).
The effect of Aldo-salt treatment on collagen content in the
myocardium is shown in Fig. 1. Aldo-salt-infused rats pre-
sented a fourfold increase (P ⬍ 0.01) in cardiac interstitial
collagen (Fig. 1B) and a threefold increase (P ⬍ 0.01) in
perivascular collagen (Fig. 1C). Animals treated with Aldo-
salt ⫹ Spiro presented similar interstitial and perivascular
collagen as control animals. As shown in Fig. 1D, molecular
analyses were also performed in cardiac tissue to study the
variations in the expression of the extracellular matrix compo-
nents, mainly collagen and collagenase activity. Aldo-salt-
infused rats presented a higher expression of ␣1-procollagen
mRNA (4-fold, P ⬍ 0.01) as well as the ratio between collagen
types I and III (5-fold, P ⬍ 0.01) and the ratio between matrix
metalloproteinase-13 and tissue inhibitor of metalloprotei-
H2375
nase-1 expression (2.3-fold, P ⬍ 0.01). Importantly, Spiro
treatment abolished Aldo-salt-induced myocardial fibrosis.
Spiro alone did not modify collagen deposition (data not
shown).
Effects of Aldo-salt treatment in myocardial inflammation in
rats. As shown in Fig. 2A, Aldo-salt infusion led to focal
inflammatory lesions. The number of focal inflammatory clus-
ters was 8 clusters/section (2 mm2) in the LV, whereas no focal
inflammatory lesions were observed in the control, Aldo ⫹
Spiro, and Spiro groups. Complementary, the number of CD3-
positive cells, a T lymphocyte marker, was higher in Aldo-salt-
treated rats compared with control rats (7-fold, P ⬍ 0.01).
Spiro treatment abolished the Aldo-salt-induced increase in
infiltrating inflammatory cells.
We also examined the effect of Aldo-salt on the expression
of plasma inflammatory markers. Aldo-salt-treated rats pre-
sented enhanced C-reactive protein (2.6-fold, P ⬍ 0.01) and
TNF-␣ (11.6-fold, P ⬍ 0.01) levels (Fig. 2B) compared with
control rats. Spiro treatment abolished the Aldo-salt-induced
increase in inflammatory markers.
Effects of Aldo-salt treatment on CT-1 expression in rats. To
localize CT-1 in the rat myocardium, paraffin-embedded sec-
tions were labeled with CT-1 antibody. High-intensity labeling
was present in cardiomyocytes and fibroblasts (Fig. 3A). Al-
though staining was generally more intense in cardiomyocytes,

Fig. 2. Inflammation in the myocardium from control, Aldo-salt, and Aldo-salt ⫹ Spiro-treated rats. A: representative photomicrograph of an Aldo-salt myocardial
Masson’s trichrome-stained section showing focal inflammatory lesions. Focal inflammatory clusters were only presented in the left ventricle (LV) of
Aldo-salt-treated rats. B: representative photomicrograph of an Aldo-salt myocardial CD3-stained section showing infiltrating inflammatory cells. C: C-reactive
protein (CRP) and TNF-␣ plasma levels were enhanced in Aldo-salt-treated animals. *P ⬍ 0.01 vs. control.

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H2376 CARDIOTROPHIN-1 MEDIATES ALDOSTERONE-INDUCED CARDIAC REMODELING

Fig. 3. Cardiotrophin (CT)-1 expression in myocardial tissue from control, Aldo-salt, and aldo-salt ⫹ Spiro-treated rats. A–C: representative cardiac sections
immunostained with CT-1 antibody. Cardiac myocytes and fibroblasts were positive for CT-1 immunostaining (A) as well as myocardial blood vessels (B). A
myocardial section a CT-1-null mouse was used as a negative control (C). D: CT-1 expression was enhanced in the myocardium from Aldo-salt-treated rats at
the protein level. Treatment with Spiro reversed Aldo-induced CT-1 expression. E: Aldo-salt-treated rats presented increased CT-1 expression at the mRNA level.
Spiro did not completely block Aldo-salt-enhanced CT-1 mRNA. *P ⬍ 0.01 vs. control.

both vascular smooth muscle cells and endothelial cells from
blood vessels throughout the cardiac tissue were also positive
for CT-1 staining (Fig. 3B). A negative control (a section from
a CT-1-null mouse) is shown in Fig. 3C. All these results were
confirmed in vitro by RT-PCR in cultured cells (not shown).
Myocardial expression of CT-1 was higher both at the
protein (Fig. 3D) and mRNA (Fig. 3E) levels (2.2- and 7.3-
fold, respectively, P ⬍ 0.01) in Aldo-salt-treated animals.
Whereas the increase in CT-1 protein expression was com-
pletely prevented by Spiro, the minerolocorticoid receptor
antagonist did not block the rise in CT-1 mRNA expression.
Spiro alone did not modify myocardial CT-1 levels (data not
shown).
Analysis of the associations in rats. Direct correlations were
found among myocardial CT-1 protein expression and hemo-
dynamic parameters, as shown in Table 3. Myocardial CT-1
expression was directly associated with SBP, DBP, MAP,
LVSP, and LVEDP.
Morever, as shown in Table 4, there were direct correlations
among myocardial CT-1 protein expression and parameters
Effects of Aldo without Na⫹ excess in general and haemo-
dynamic parameters in WT and CT-1-null mice. As shown in
Table 5, BW was similar in WT and CT-1-null mice treated
with vehicle or Aldo. Of note, HW was higher (P ⬍ 0.05) in
both WT and CT-1-null mice treated with Aldo than in their
respective control group. However, only WT mice infused with
Aldo presented an increase (P ⬍ 0.05) in HW/BW, indicating
cardiac hypertrophy. Aldo failed to increase blood pressure
levels in WT and CT-1-null mice, and the four experimental
groups of animals had similar values of SBP, DBP, MAP, and
HR. HW and HW/BW were slightly increased (6% and 5%,
respectively, P ⬍ 0.05) in control CT-1-null mice compared
with control WT mice.
Table 3. Associations found between myocardial CT-1
expression and hemodynamic parameters
CT-1 Protein

assessing LVH (i.e., LVCSA and cardiomyocyte dimensions) r P value

and fibrosis (i.e., collagen type I-to-III ratio and interstitial SBP, mmHg 0.685 0.001
fibrosis) in all treated rats. Multiple linear regression analysis DBP, mmHg 0.646 0.007
demonstrated that, when adjusted for confounding factors (i.e., MAP, mmHg 0.623 0.003
blood pressure levels), the direct associations between CT-1 LVEDP, mmHg 0.549 0.040
LVSP, mmHg 0.733 0.002
and cardiomyocyte length and width, ␣1-procollagen, collagen
type I-to-III ratio, and interstitial collagen remained significant. CT-1, cardiotrophin-1.

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 CARDIOTROPHIN-1 MEDIATES ALDOSTERONE-INDUCED CARDIAC REMODELING H2377
Table 4. Associations found between myocardial CT-1 Table 6. Histological and molecular parameters assessing myo-
expression and parameters assessing myocardial remodeling cardial remodeling in WT and CT-1-null mice infused with Aldo
CT-1 Protein WT Mice CT-1-Null Mice

r P value Control Aldo Control Aldo

LVCSA, mm2 0.549 0.034 n 8 8 8 8

LVWT, mm 0.497 0.045 LVCSA, mm2 13.5 ⫾ 0.2 15.0 ⫾ 0.2† 14.8 ⫾ 0.7 14.8 ⫾ 0.5
Cardiomyocyte length, ␮m 0.539 0.026 LVWT, mm 1.5 ⫾ 0.1 1.7 ⫾ 0.2† 1.6 ⫾ 0.1 1.7 ⫾ 0.1
Cardiomyocyte width, ␮m 0.713 0.001 Cardiomyocyte
c-Fos protein 0.645 0.004 width, ␮m 15.1 ⫾ 0.3 16.1 ⫾ 0.3† 15.2 ⫾ 0.6 15.2 ⫾ 0.5
c-Myc protein 0.621 0.006 c-Fos protein, AU 1.12 ⫾ 0.4 2.64 ⫾ 0.3* 0.14 ⫾ 0.05 0.21 ⫾ 0.1†
␣1-Procollagen mRNA 0.893 0.001 c-Myc protein, AU 0.47 ⫾ 0.1 1.15 ⫾ 0.2* 0.33 ⫾ 0.07 0.62 ⫾ 0.09†
Collagen type I/collagen type III, AU 0.542 0.036
MMP-13/TIMP-1, AU 0.522 0.026 Values are means ⫾ SE; n, no. of animals. *P ⬍ 0.01 vs. control; †P ⬍ 0.05
Interstitial collagen, % 0.624 0.003 vs. control.
Perivascular collagen, % 0.568 0.036
MMP-13, matrix metalloproteinase-13; TIMP-1, tissue inhibitor of metal- 0.05). However, in mice lacking CT-1, Aldo infusion increased
loproteinase-1. only ␣1-procollagen mRNA (80%, P ⬍ 0.05), without modi-
fying the the collagen type I-to-III ratio. The effects of Aldo on
Effects of Aldo without Na⫹ excess in myocardial remodel- interstitial fibrosis and ␣1-procollagen mRNA expression were
ing in WT and CT-1-null mice. Cardiac morphometric and significantly different in WT mice compared with CT-1-null
molecular analyses are shown in Table 6. WT mice infused mice (P ⬍ 0.05).
with Aldo presented increased LVCSA (11%, P ⬍ 0.05) and Effects of Aldo without Na⫹ excess in myocardial inflam-
augmented LVWT (13%, P ⬍ 0.05) that were accompanied by mation in WT and CT-1-null mice. As shown in Fig. 5A, Aldo
an increase in cardiomyocyte width (6%, P ⬍ 0.05). On the treatment caused the appearance of more focal inflammatory
other hand, Aldo failed to induce histological modifications in lesions in WT mice than in CT-1-null mice. The number of
hearts from mice lacking CT-1 (representative photographs are focal inflammatory clusters in WT mice was 3 clusters/section
shown in Fig. 3A). LVCSA and LVWT were slightly increased (0.75 mm2) in the LV, whereas in CT-1-null mice the number
(9% and 6%, respectively, P ⬍ 0.05) in control CT-1-null mice was 2 clusters/section (0.4 mm2). No focal inflammatory le-
compared with control WT mice. At the molecular level, Aldo sions were observed in the control groups of WT and CT-1-null
treatment increased myocardial expression of c-fos (2.4-fold, mice. Complementary, CD3 immunostaining revealed that in
P ⬍ 0.01) and c-myc (2.5-fold, P ⬍ 0.01) in WT mice, whereas response to Aldo, WT mice accumulated more CD3-positive
in CT-1-null mice, the increases were significantly lower cells (345%) than CT-1-null mice (265%). The effects of Aldo
(c-fos: 50%, P ⬍ 0.05, and c-myc: 88%, P ⬍ 0.05). c-fos and on the number of focal inflammatory lesions and CD45 immu-
c-myc expressions were decreased (8-fold, P ⬍ 0.01, and 40%, nostaining were significantly different in WT mice compared
P ⬍ 0.05, respectively) in control CT-1-null mice compared with CT-1-null mice (P ⬍ 0.05).
with control WT mice. Both WT and CT-1-null mice treated with Aldo presented
The effect of Aldo treatment on collagen content in the enhanced plasma C-reactive protein (90% and 88%, P ⬍ 0.01,
myocardium from WT and CT-1-null mice is shown in Fig. 4. respectively). However, plasma TNF-␣ expression was only
Aldo-infused WT mice presented a 250% increase (P ⬍ 0.01) augmented by Aldo in WT mice (75%, P ⬍ 0.01; Fig. 5B).
in cardiac interstitial collagen, whereas Aldo-infused CT-1- Effects of Aldo without Na⫹ excess on CT-1 expression in
null mice only presented a 70% increase (P ⬍ 0.05; Fig. 4B). WT mice. Myocardial expression of CT-1 were higher (2.3-
Aldo induced a 166% increase (P ⬍ 0.01) in perivascular fold, P ⬍ 0.01) in normotensive Aldo-treated WT mice com-
collagen in WT mice, whereas the mineralocorticoid failed to pared with control mice, as shown in Fig. 6.
augment perivascular collagen in CT-1-null mice (Fig. 4C). As DISCUSSION
shown in Fig. 4D, Aldo-infused WT mice exhibited a higher
expression of ␣1-procollagen mRNA (2.5-fold, P ⬍ 0.01) as The purpose of this study was to investigate the role of CT-1
well as the ratio between collagen types I and III (1.7-fold, P ⬍ in cardiovascular remodeling induced by Aldo in rodents.

Table 5. General and hemodynamic parameters in WT and CT-1-null mice infused with Aldo
WT Mice CT-1-Null Mice

Control Aldo Control Aldo

n 8 8 8 8
BW, g 28.7 ⫾ 0.6 28.1 ⫾ 0.5 28.7 ⫾ 0.4 29.7 ⫾ 0.5
HW, g 0.157 ⫾ 0.004 0.168 ⫾ 0.004* 0.166 ⫾ 0.006 0.172 ⫾ 0.007*
HW/BW, mg/g 5.49 ⫾ 0.2 5.99 ⫾ 0.1* 5.79 ⫾ 0.2 5.82 ⫾ 0.3
SBP, mmHg 122 ⫾ 2 123 ⫾ 4 114 ⫾ 2 116 ⫾ 3
DBP, mmHg 101 ⫾ 1 102 ⫾ 4 101 ⫾ 2 92 ⫾ 3
MAP, mmHg 112 ⫾ 2 112 ⫾ 4 108 ⫾ 2 104 ⫾ 3
HR, beats/min 627 ⫾ 12 630 ⫾ 20 606 ⫾ 23 606 ⫾ 22
Values are means ⫾ SE; n, no. of animals. WT, wild type. *P ⬍ 0.05 vs. control.

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H2378 CARDIOTROPHIN-1 MEDIATES ALDOSTERONE-INDUCED CARDIAC REMODELING

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 CARDIOTROPHIN-1 MEDIATES ALDOSTERONE-INDUCED CARDIAC REMODELING
Here, we demonstrate, for the first time, that Aldo induced
CT-1 expression in vivo, independent from blood pressure
levels. Moreover, the increased CT-1 was associated with
hemodynamic parameters and with parameters showing LVH
and fibrosis. The data obtained in CT-1-null mice clearly
demonstrate that CT-1 is a key mediator in Aldo-induced
harmful myocardial effects.
Numerous studies have documented that increased Aldo, in
addition to excess dietary salt and the removal of one kidney,
elevates blood pressure and induces LVH, fibrosis, inflamma-
tory responses, and dysfunction (3, 15, 16, 18, 20, 28) and that
the effects of Aldo are crucially dependent on the salt status of
the rat (2). On the other hand, it has been described that excess
Aldo under a normal salt diet, with minor blood pressure
elevation, plays a pivotal role in cardiac remodeling and
inflammation (34). Therefore, in the present study, two differ-
ent models were used: rats treated with Aldo-salt, which
presented elevated arterial pressure, and mice treated with
Aldo not subjected to Na⫹ loading, which presented normal
blood pressure levels. In normal nonuninephrectomized rats,
Aldo-salt infusion increased blood pressure and also induced
diastolic dysfunction accompanied by LVH, fibrosis, and in-
flammation, as previously reported (3, 15, 16, 18, 20, 28). The
mineralocorticoid receptor blocker Spiro inhibited Aldo-in-
duced high blood pressure and myocardial remodeling. These
data are in accordance with results showing that in experimen-
tal models, blockade of the mineralocorticoid receptor im-
proved not only LVH and fibrosis but also diastolic dysfunc-
tion (6). Furthermore, Spiro treatment improved myocardial
inflammation, zinc dyshomeostasis, and oxidative strress (28,
30, 34). Finally, it is important to point out that mineralocor-
ticoid receptor blockade improves outcome and myocardial
remodeling in patients with HF (22, 36, 37). In WT mice
subjected to the same dose of Aldo, the mineralocorticoid
failed to induce blood pressure modifications, but it induced
LVH, fibrosis, and inflammatory lesions. The fact that Aldo did
not cause hypertension but induced LVH, fibrosis, and inflam-
mation in mice suggests that it has direct cardiac effects
independent of changes in blood pressure. Our data differed
from previous findings in regard to the slight increase in blood
pressure levels induced by Aldo without Na⫹ excess in rats
(34). The difference between our present observations and
previous findings could be related to the fact that the mice were
not uninephrectomized and the fact that C57BL6 mice seem to
be resistant to hypertensive stimuli (7, 26, 32, 33).
Molecular mechanisms underlying Aldo-induced LVH and
fibrosis have to be delineated. Previous studies have suggested
the involvement of ANG II (8), oxidative stress (28, 34),
magnesium (26), apoptosis signal-regulating kinase-1 (17),
transforming growth factor-␤ (15), or connective tissue growth
factor (15). We (9, 10) have previously published that Aldo
H2379
induced the expression of CT-1, a profibrotic and hypertrophic
cytokine, in both adult cardiomyocytes and vascular cells,
suggesting a role for the cytokine in Aldo-induced cardiomy-
ocyte hypertrophy and vascular alterations. In the present
study, we found that Aldo-salt hypertensive rats, as well as
normotensive Aldo-treated mice, exhibited increased myocar-
dial CT-1, and the cytokine was associated with parameters
showing myocardial remodeling independent of blood pressure
levels. Moreover, CT-1 was associated with LV pressure,
suggesting that the cytokine is produced as a response to
mechanical stress presented in the myocardial wall. The fact
that CT-1 is expressed in cardiac myocytes, fibroblasts, and
vascular cells reinforces the associations found between the
cytokine and Aldo-induced effects on hemodynamics, hyper-
trophy, and interstitial and perivascular fibrosis. Related to this,
previous studies (11, 14, 19) have reported CT-1 elevations in
both experimental models and patients with hypertension.
Moreover, in HF patients, high Aldo levels were associated
with high CT-1 levels (12). Therefore, the possible role of
hemodynamic factors in CT-1 induction remains uncertain.
However, CT-1 was also increased in the myocardium from
normotensive WT mice infused with Aldo, indicating that
CT-1 induction by Aldo is independent of hemodynamic fac-
tors. Finally, we demonstrated that CT-1 is a necessary factor
for the accumulation of fibrous tissue and the development of
LVH induced by Aldo. Indeed, Aldo failed to induce LVH and
fibrosis in mice lacking CT-1, suggesting that the absence of
the cytokine blocked the Aldo-induced effects on cardiac
remodeling. Moreover, in the absence of CT-1, Aldo proin-
flammatory effects were also diminished. Thus, our present
findings provide a new molecular mechanism underlying the
cardiac remodeling and injury caused by Aldo in vivo and
independent of hemodynamic changes.
In conclusion, our present findings provide the first evidence
that CT-1, independent of hypertension, significantly partici-
pates in Aldo-induced LVH and fibrosis. Therefore, we suggest
that CT-1 could be a new biotarget to reduce the cardiac
remodeling induced by Aldo in cardiovascular diseases.
ACKNOWLEDGEMENTS
The authors specially thank Pr. Simon Thornton for editing this manuscript
and Sandra Ballesteros for technical assistance.
GRANTS
This work was supported by grants from the Foundation for Applied
Medical Research, Institut National de la Santé et de la Recherche Médicale,
Fondo de Investigaciones Sanitarias (PS09/0871) and Red Temática de Inves-
tigación Cardiovascular RECAVA (RD06/0014/007 and RD06/0014/008)
from the Instituto de Salud Carlos III, Plan Nacional de Investigación y
Desarrollo (MICINN, Spain), Project: SAF2007-61595, by the Programme
National de Recherche Cardiovasculaire, La-Société Française d’Hypertension
artérielle, la Région Lorraine, the 6th EU-FP Network of Excellence “Inge-
nious HyperCare” (LSHM-CT-2006-037093).

Fig. 4. Morphology and composition of the myocardium from wild-type (WT) and CT-1-null mice infused with Aldo without Na⫹ excess. A: representative
photomicrographs of myocardial Masson’s trichrome-stained sections from WT and CT-1-null mice treated with vehicle or Aldo. B: interstital myocardial fibrosis
was evaluated in Sirius red-stained sections. After 3 wk of treatment, Aldo increased interstitial collagen deposition in WT mice, whereas the increase was
significantly lower in CT-1-null mice. The effect of Aldo on interstitial fibrosis was significantly higher in WT mice compared with CT-1-null mice.
C: perivascular myocardial fibrosis was quantified in sections stained with Masson’s trichrome. Aldo augmented perivascular fibrosis in WT mice. CT-1-null mice
were resistant to Aldo-induced perivascular collagen accumulation. D: the expression of extracellular matrix components was quantified by RT-PCR and Western
blot analysis. Aldo treatment increased ␣1-procollagen and the collagen type I-to-III ratio in WT mice. The effect of Aldo on ␣1-procollagen was significantly
higher in WT mice compared with CT-1-null mice. *P ⬍ 0.05 vs. control (same strain); $P ⬍ 0.05 vs. WT mice (same treatment).

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H2380 CARDIOTROPHIN-1 MEDIATES ALDOSTERONE-INDUCED CARDIAC REMODELING

Fig. 5. Inflammation in the myocardium from WT and CT-1-null mice infused with Aldo without Na⫹ excess. A: representative photomicrographs of Aldo-treated
myocardial Masson’s trichrome-stained sections from WT and CT-1-null mice showing focal inflammatory lesions. Aldo-treated WT mice presented more and
larger focal inflammatory lesions than Aldo-treated CT-1-null mice. B: representative photomicrographs of Aldo-treated myocardial CD3-stained sections from
WT and CT-1-null mice demonstrating infiltrating inflammatory cells. Aldo-treated WT mice presented higher immunostaining than Aldo-treated CT-1-null mice.
C: CRP plasma levels were enhanced in both WT and CT-1-null mice treated with Aldo, whereas TNF-␣ plasma levels were enhanced only in Aldo-treated WT
mice. *P ⬍ 0.05 vs. control (same strain); $P ⬍ 0.05 vs. WT mice (same treatment).

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 CARDIOTROPHIN-1 MEDIATES ALDOSTERONE-INDUCED CARDIAC REMODELING
Fig. 6. CT-1 expression in myocardial tissue from control mice treated with
Aldo without Na⫹ excess. CT-1 expression was quantified by ELISA. Aldo
treatment enhanced myocardial CT-1 expression in WT mice compared with
control mice. *P ⬍ 0.01 vs. control.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: N.L.-A., P.R., F.Z., V.L., M.A.F., V.C., and J.D.
conception and design of research; N.L.-A. and B.M.-F. performed experi-
ments; N.L.-A. and B.M.-F. analyzed data; N.L.-A., B.M.-F., V.L., M.A.F.,
V.C., and J.D. interpreted results of experiments; N.L.-A., M.A.F., and V.C.
prepared figures; N.L.-A., P.R., F.Z., V.L., V.C., and J.D. drafted manuscript;
N.L.-A., P.R., F.Z., V.L., V.C., and J.D. edited and revised manuscript;
N.L.-A., B.M.-F., P.R., F.Z., V.L., M.A.F., V.C., and J.D. approved final
version of manuscript.
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