Na+/H+ exchange inhibition attenuates left ventricular remodeling and preserves systolic function in pressure-overloaded hearts.
ABSTRACT Cardiac hypertrophy is a homeostatic response to elevated afterload. Na+/H+ exchanger (NHE) inhibition reduces the hypertrophic response in animal models of left ventricular hypertrophy (LVH) and myocardial infarction. We examined the effect of chronic treatment with cariporide, a selective inhibitor of Na+/H+ exchanger isoform 1 (NHE-1), on left ventricular (LV) systolic and diastolic function under pressure overload conditions. Male CD-1 mice were randomized to receive either a control diet or an identical diet supplemented with 6000 p.p.m. of cariporide. Cardiac pressure overload was induced by thoracic aortic banding. LV dimension and systolic and diastolic function were assessed in sham and banded mice by echocardiography and cardiac catheterization 2 and 5 weeks after surgery. Histological analysis was also performed. After 2 weeks of pressure overload, the vehicle-treated banded mice (Veh-Bd) had enhanced normalized LV weight (about +50%) and normal chamber size and function, whereas cariporide-treated banded mice (Car-Bd) showed a preserved contractility and systolic function despite a marked attenuation of LVH. Diastolic function did not differ significantly among groups. After 5 weeks, the Veh-Bd developed LV chamber enlargement and systolic dysfunction as evidenced by a 16% increase in LV end-diastolic diameter, a 36% decrease in myocardial contractility, and a 26% reduction in percent fractional shortening. In contrast, Car-Bd showed an attenuated increase in LV mass, normal chamber size, and a maintained systolic function. A distinct histological feature was that in banded mice, cariporide attenuated the development of cardiomyocyte hypertrophy but not the attendant myocardial fibrosis. In conclusion, the results of the present study indicate that (i) the hypertrophic response to pressure overload is dependent on NHE-1 activity, and (ii) at the 5-week stage, banding-induced deterioration of LV performance is prevented by NHE-1 inhibition.British Journal of Pharmacology (2004) 141, 526-532. doi:10.1038/sj.bjp.0705631
- SourceAvailable from: William Kutschke[show abstract] [hide abstract]
ABSTRACT: Cardiac hypertrophy is considered a necessary compensatory response to sustained elevations of left ventricular (LV) wall stress. To test this, we inhibited calcineurin with cyclosporine (CsA) in the setting of surgically induced pressure overload in mice and examined in vivo parameters of ventricular volume and function using echocardiography. Normalized heart mass increased 45% by 5 weeks after thoracic aortic banding (TAB; heart weight/body weight, 8.3+/-0.9 mg/g [mean+/-SEM] versus 5. 7+/-0.1 mg/g unbanded, P<0.05). Similar increases were documented in the cell-surface area of isolated LV myocytes. In mice subjected to TAB+CsA treatment, we observed complete inhibition of hypertrophy (heart weight/body weight, 5.2+/-0.3 mg/g at 5 weeks) and myocyte surface area (endocardial and epicardial fractions). The mice tolerated abolition of hypertrophy with no signs of cardiovascular compromise, and 5-week mortality was not different from that of banded mice injected with vehicle (TAB+Veh). Despite abolition of hypertrophy by CsA (LV mass by echo, 83+/-5 mg versus 83+/-2 mg unbanded), chamber size (end-diastolic volume, 33+/-6 microL versus 37+/-1 microL unbanded), and systolic ejection performance (ejection fraction, 97+/-2% versus 97+/-1% unbanded) were normal. LV mass differed significantly in TAB+Veh animals (103+/-5 mg, P<0.05), but chamber volume (end-diastolic volume, 44+/-6 microL), ejection fraction (92+/-2%), and transstenotic pressure gradients (70+/-14 mm Hg in TAB+Veh versus 77+/-11 mm Hg in TAB+CsA) were not different. In this experimental setting, calcineurin blockade with CsA prevented LV hypertrophy due to pressure overload. TAB mice treated with CsA maintain normal LV size and systolic function.Circulation 06/2000; 101(24):2863-9. · 15.20 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: S.-I. Takewaki, M. Kuro-o, Y. Hiroi, T. Yamazaki, T. Noguchi, A. Miyagishi, K.-I. Nakahara, M. Aikawa, I. Manabe, Y. Yazaki and R. Nagai, Activation of Na+-H+ Antiporter (NHE-1) Gene Expression during Growth, Hypertrophy and Proliferation of the Rabbit Cardiovascular System, Journal of Molecular and Cellular Cardiology (1995) 27, 729-742, The Na+-H+ antiporter is a unique transmembrane protein with multiple roles in cellular functions through intracellular alkalization. It participates in the regulation of intracellular pH, cell volume and intracellular signalling in response to various mitogenic stimuli. To clarify its role as a subcellular signal in cardiovascular remodeling like vascular hyperplasia or cardiac hypertrophy, we determined mRNA levels of the Na+-H+ antiporter isoform, NHE-1, in vascular smooth muscles and pressure-overloaded hearts in rabbits. The NHE-1 mRNA levels in rabbit aortas and hearts were de-velopmentally regulated with high levels at embryonic and neonatal stages than in adults. In primary-cultured smooth muscle cells (SMC), the mRNA levels were increased during exponential growth, but decreased to initial levels at coniuency. Growth of a mutant SMC line, C5, which is deficient in Na+-H+ antiporter activity, was markedly reduced in bicarbonate-free medium. However, when the activity was restored by transfecting cells with a full-length NHE-1 cDNA in an expression vector, the growth rate of C5 was accelerated again. After balloon injury to the vascular wall, the NHE-1 mRNA levels of the injured arteries were also increased, suggesting that Na+-H+ antiporter contributes to the network of the growth promoting systems in smooth muscle cells in vivo. Pressure-overload on the ventricle increased the NHE-1 mRNA levels in hearts approximately two-fold of sham-operated rabbits after 3 days and remained for at least; two weeks (P<0.05). We further demonstrated that 3-m.ethylsulfonyl-4-piperidino-benzoyl guanidine mesylate (Hoe 694), a potent antagonist of Na+-H+ antiporter, partially inhibited stretch-induced activation of mitogen-activated kinase (MAP kinase) in the cultured cardiomyocytes. From these results, we conclude that activation of the Na+-H+ antiporter and its gene expression is involved in molecular mechanisms of both cardiac hypertrophy and vascular smooth muscle cell proliferation, indicating a potential target in developing new therapeutics for cardiovascular diseases.Journal of Molecular and Cellular Cardiology 01/1995; · 5.15 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Intracellular free sodium levels ([Na+]i) were assessed with 23Na nuclear magnetic resonance (NMR) spectroscopy in isolated, Langendorff-perfused normal, compensated hypertrophied, and hypertrophied failing guinea pig hearts under several conditions. Baseline [Na+]i measured with a shift reagent was significantly greater than normal in the compensated hypertrophied hearts (12.8 +/- 1.2 mmol/L v 6.4 +/- 0.7 mmol/L, means +/- SEM, P < .01), but not in the hypertrophied failing hearts (8.7 +/- 1.9 mmol/L, P = N.S.). Moreover, the highest levels of [Na+]i were seen just 3 to 4 weeks after aortic constriction. [Na+]i was inversely related to both time after aortic constriction (R = -0.71, P < .03) and to the degree of left ventricular hypertrophy (R = -0.79, P < .01), suggesting that the hypertrophied failing heart is capable of maintaining relatively normal [Na+]i. In addition, triple quantum filtered NMR measurements were made to assess changes in [Na+]i subsequent to altered perfusion or loading conditions. In hypertrophied failing hearts, but not normal hearts, low coronary perfusion pressure (60 cm H2O) was associated with relatively higher [Na+]i (ANOVA, P < .05), suggesting greater sensitivity of hypertrophied failing hearts to hypoperfusion. On the other hand, when all hearts were perfused at 90 cm H2O and intraventricular balloon volume was increased from 100 microL to 300 microL, [Na+]i increased significantly only in the normal guinea pig hearts (12.3 +/- 1.8%, P < .01). These findings suggest complex changes in the expression or modulation of proteins involved in Na+ regulation. Interpretation regarding the physiological significance of these changes depends on the specific mechanism(s) proposed. Previous work in this and other models of hypertrophy suggest that changes in the number or activity of both Na(+)-K(+)-ATPase and Na(+)-Ca2+ exchange proteins are involved.American Journal of Hypertension 10/1995; 8(9):934-43. · 3.67 Impact Factor
Naþ/Hþexchange inhibition attenuates left ventricular
remodeling and preserves systolic function in pressure-overloaded
*,1Giuseppe Marano,2Alessandro Vergari,3Liviana Catalano,1Simona Gaudi,1Sergio Palazzesi,
1Marco Musumeci,1Tonino Stati &4Alberto U. Ferrari
1Laboratorio di Farmacologia, Istituto Superiore di Sanita ` , Viale Regina Elena 299, Rome, Italy;2Facolta ` di Medicina e
Chirurgia, Istituto di Anestesia e Rianimazione, Universita ` Cattolica del Sacro Cuore, Rome, Italy;3Laboratorio di Biochimica
Clinica, Istituto Superiore di Sanita ` , Rome, Italy and4Centro di Fisiologia Clinica e Ipertensione, University of Milano-Bicocca,
inhibition reduces the hypertrophic response in animal models of left ventricular hypertrophy (LVH)
and myocardial infarction. We examined the effect of chronic treatment with cariporide, a selective
inhibitor of Naþ/Hþexchanger isoform 1 (NHE-1), on left ventricular (LV) systolic and diastolic
function under pressure overload conditions.
2Male CD-1 mice were randomized to receive either a control diet or an identical diet supplemented
with 6000p.p.m. of cariporide. Cardiac pressure overload was induced by thoracic aortic banding. LV
dimension and systolic and diastolic function were assessed in sham and banded mice by
echocardiography and cardiac catheterization 2 and 5 weeks after surgery. Histological analysis
was also performed.
3After 2 weeks of pressure overload, the vehicle-treated banded mice (Veh-Bd) had enhanced
normalized LV weight (about þ50%) and normal chamber size and function, whereas cariporide-
treated banded mice (Car-Bd) showed a preserved contractility and systolic function despite a marked
attenuation of LVH. Diastolic function did not differ significantly among groups. After 5 weeks, the
Veh-Bd developed LV chamber enlargement and systolic dysfunction as evidenced by a 16% increase
in LV end-diastolic diameter, a 36% decrease in myocardial contractility, and a 26% reduction in
percent fractional shortening. In contrast, Car-Bd showed an attenuated increase in LV mass, normal
chamber size, and a maintained systolic function. A distinct histological feature was that in banded
mice, cariporide attenuated the development of cardiomyocyte hypertrophy but not the attendant
4 In conclusion, the results of the present study indicate that (i) the hypertrophic response to
pressure overload is dependent on NHE-1 activity, and (ii) at the 5-week stage, banding-induced
deterioration of LV performance is prevented by NHE-1 inhibition.
British Journal of Pharmacology (2004) 141, 526–532. doi:10.1038/sj.bjp.0705631
Pressure overload; Naþ/Hþexchanger; cardiac remodeling; systolic function
Cardiac hypertrophy is a homeostatic response to elevated afterload. Naþ/Hþexchanger (NHE)
Abbreviations: BW, body weight; Car, cariporide; Car-Bd, cariporide-treated, banded mice; Car-Sh, cariporide-treated, sham-
operated mice; CSA, cardiomyocyte cross-sectional area; Ees, end-systolic elastance; HR, heart rate; LVEDD, left
ventricular end-diastolic diameter; LVEDP, left ventricular end-diastolic pressure; LV FS, left ventricular
fractional shortening; LVSP, left ventricular systolic pressure; LVW/BW, left ventricular weight-to-body weight
ratio; NHE-1, Naþ/Hþexchanger isoform 1; RWT, posterior-wall thickness-to-left ventricular end-diastolic
diameter ratio; ss, end-systolic left ventricular wall stress; Veh-Sh, vehicle-treated, sham-operated mice; Veh-Bd,
vehicle-treated, banded mice
Hypertension-related left ventricular hypertrophy (LVH)
strongly enhances the risk of future cardiovascular events
and death, so that preventing LVH is a major goal in the
clinical management of hypertension.
Previous studies have indicated that cardiac hypertrophy is
associated with an increase in intracellular sodium (Na[i])
(Jelicks & Siri, 1995; Gray et al., 2001; Pogwizd et al., 2003;
Verdonck et al., 2003). Conversely, an increase in Na[i]
(produced by, e.g. inhibiting Naþ/Kþ-ATPase with ouabain
or enhancing extracellular sodium concentration) can promote
cardiomyocyte hypertrophy (Peng et al., 1996; Gu et al., 1998).
Furthermore, a recent study from our laboratory showed that
agents interfering with fast sodium channel function (e.g. d-
propranolol or disopyramide) display antihypertrophic activ-
ity in pressure-overloaded rat hearts (Marano et al., 2002).
*Author for correspondence; E-mail: firstname.lastname@example.org
Advance online publication: 12 January 2004
British Journal of Pharmacology (2004) 141, 526–532
& 2004 Nature Publishing GroupAll rights reserved 0007–1188/04 $25.00
Taken together, these observations suggest that the mechan-
isms regulating cellular sodium homeostasis are involved in the
cardiac hypertrophic response secondary to pressure overload.
The cardiac sodium/hydrogen exchanger, in addition to its
role in intracellular pH regulation, is a pathway for Naþinflux
(Frelin et al., 1984) and its pharmacological blockade should
promote a decrease of Na[i] levels. The activation of the
cardiac sodium ion/hydrogen ion exchanger isoform 1 (NHE-1)
has been implicated in the cardiomyocyte hypertrophic
process, inasmuch as (a) in pressure-overloaded hearts, both
NHE-1 mRNA levels and Na[i] concentration are significantly
elevated compared to control, nonoverloaded hearts (Take-
waki et al., 1995), (b) the activity of the antiporter is
augmented by hypertrophic factors such as a1-adrenergic
activation (Yokoyama et al., 1998) and angiotensin II
(Gunasegaram et al., 1999), and (c) cariporide, a newly
developed NHE-1-specific inhibitor (Scholz et al., 1995),
inhibits the hypertrophy of cardiac myocytes in DOCA/salt
rats (Fujisawa et al., 2003) and in response to stretch as well as
to b1-adrenergic receptor (b1-AR) stimulation (Yamazaki et al.,
1998; Engelhardt et al., 2002). Additionally, it has been shown
that cariporide was able (i) to attenuate compensatory right
ventricular hypertrophy following pulmonary vascular injury
(Chen et al., 2001), (ii) to inhibit the development of
pathological remodeling after myocardial infarction (Kusu-
moto et al., 2001), and (iii) to regress both cardiomyocyte
hypertrophy and interstitial fibrosis in the spontaneously
hypertensive rats (Camilion De Hurtado et al., 2002; Cingolani
et al., 2003). Suppression of the compensatory myocardial
hypertrophy might be expected to induce systolic dysfunction
more readily because chronic pressure overload-induced LVH
is thought to develop as an adaptive response by which LV
wall stress is moderated and LV function is supported.
However, the consequences of NHE-1 inhibition on systolic
function and cardiac contractility in pressure-overloaded
animals have not been investigated.
Therefore, the aim of the present study was to evaluate the
effect of chronic treatment with cariporide on systolic and
diastolic LV function and remodeling after thoracic aortic
banding (TAB). This is a standard maneuver that reproducibly
induces LV pressure overload, development of LVH and
transition to LV dilation and dysfunction within a few weeks
after surgery (Esposito et al., 2002), in adult mice.
CD-1 mice (12-week-old male, 28–30g) were shipped by
Charles River (Calco, Como, Italy) and maintained in
accordance with EC guidelines (86/609/EEC) and with the
Guide for the Care and Use of Laboratory Animals (NIH 85-23,
revised 1996). Mice were randomly assigned to four groups:
sham surgery plus control diet; sham surgery plus cariporide
diet (containing 6000p.p.m. of cariporide); aortic banding (see
below) plus control diet; aortic banding plus cariporide diet.
Animals were maintained for 2 and 5 weeks after surgery
and were then anesthetized with isoflurane in order to
allocated to cariporide treatment, the drug was administered
starting 10 days before banding surgery to allow plasma
levels to reach steady-state values (Kusumoto et al., 2001).
Although circulating cariporide levels were not determined
in our study, it was shown (Engelhardt et al., 2002) that
mice treated with a dosage similar to ours had cariporide
levels of 2.570.3mmoll–1, able to inhibit NHE-1 activity
by more than 50% in isolated cardiomyocytes (Scholz et al.,
hemodynamic measurements. Inthe animals
Mouse model of LV pressure overload
The development of LVH was induced by banding the thoracic
aorta. Each mouse was anesthetized with a mixture of
ketamine (100mgkg–1) and xylazine (5mgkg–1), orally intu-
bated with a 24-gauge tubing under direct laryngeal visualiza-
tion, and ventilated with a tidal volume of 200ml and a
respiratory rate of 110breathsmin–1. The chest was then
opened at the second intercostal space. TAB was performed by
placing a ligature on the thoracic aorta between the
innominate artery and left common carotid artery along with
a 25-gauge needle using a 6-0 nylon suture. After the suture
was secured, the needle was removed, the chest was closed,
and the pneumothorax evacuated. The mortality rate for
TAB was 3.5% (4/114) during the surgical procedure
and 14.5% (16/110) after 24h. The perioperative deaths
could be attributed to aortic rupture or acute heart failure.
A concurrent group of mice was subjected to a sham
operation, in which an identical surgical procedure was
performed but the ligature was not tightened. The total
perioperative mortality was 0% for sham-operated mice. To
measure pressure aortic gradient, both right- and left-carotid
arteries were cannulated by a 1.4-F, four-electrode pressure–
volume Millar catheter and stretched P50 tubing, respectively.
The Millar catheter was connected to the pressure–conduc-
tance unit (model MPCU-200, Millar, U.S.A.), whereas the
other catheter was connected to a P23 Statham pressure
At 2 and 5 weeks after banding, hemodynamic analyses were
performed in mice intubated and anesthetized with isoflurane
(1% in 100% oxygen). Echocardiographic examination was
performed with an Esaote SIM 7000 Challenge (Esaote
Biomedica, Firenze, Italy) equipped with a 10MHz imaging
transducer. After good-quality 2D short-axis images of the left
ventricle were obtained, M-mode freeze frames were printed
on common echocardiographic paper and digitized. End-
systolic (left ventricular end-systolic diameter, LVESD) and
end-diastolic (left ventricular end-diastolic diameter, LVEDD)
left ventricular internal diameters, and posterior-wall end-
diastolic (PWTd) and end-systolic (PWTS) thickness were
measured by an image-analysis system (Metamorph, Universal
Image Corporation, PA, U.S.A.). Percent fractional short-
ening was calculated as
ðLVEDD ? LVESDÞ
G. Marano et al
LV function and myocardial NHE-1 inhibition527
British Journal of Pharmacology vol 141 (3)
End-systolic wall stress was estimated as follows:
ð4PWTSÞ 1 þ
where Psis the peak left ventricular systolic pressure (LVSP)
(in mmHg) and 1.35 is the conversion factor from mmHg to
grams per centimeter squared. LVESD and PWTs were
measured from the M-mode echocardiogram. These measure-
ments were made at end-systole, as defined by the time point
corresponding to the smallest cavity dimension.
To measure arterial blood pressure and to obtain LV pressure–
volume curves, a 1.4-F, four-electrode pressure–volume
catheter (model SPR-839, Millar Instruments, U.S.A.) was
inserted into the right carotid artery, and retrogradely
advanced through the ascending aorta into the LV under
isoflurane anesthesia. The pressure–volume catheter was
connected to an MPCU-200 unit (Millar Instruments,
U.S.A.). Correct catheter positioning was confirmed by online
visualization of the pressure–volume loops. Conductance-
derived pressure–volume data were analyzed with IOX soft-
ware (version 1594, EMKA, France). The end-systolic point of
pressure–volume loops was computed by an iterative method
and end-systolic elastance (Ees) was determined from end-
systolic pressure–volume relationships (ESPVRs). To change
the cardiac preload, inferior caval occlusion was produced
over 3s. Opening the thorax induced a drop in LV pressure in
all groups. All steady-state and caval occlusion pressure–
volume loops were acquired, while pulmonary ventilation was
temporarily suspended. The data were recorded as a series of
8–10 pressure–volume loops.
At the end of the in vivo experiment, animals were deeply
anesthetized, the heart was removed and the left ventricle was
separated from the other chambers, weighed and normalized
by body weight (BW) to determine LVH. For histological
assessment, the LV was fixed with 10% buffered formalin
solution and embedded in paraffin. LV sections, 4mm thick,
were cut and stained with hematoxylin and eosin for the
measurement of myocyte cross-sectional area or by the sirius
red/picric acid method to determine LV fibrosis by quantita-
tive morphometry (Morphometric, Universal Imaging Cor-
poration, PA, U.S.A.). Myocyte mean cross-sectional area was
calculated by measuring the dimensions of no less than 50 cells
per section (four or five sections of each left ventricle). The
visual fields were accepted for quantitative analysis if cross-
sections of myocytes were present, nuclei were visible, and
their cellular membranes were intact. Cardiomyocyte width
was assessed by marking the borders of the cells. The extent of
interstitial LV fibrosis was calculated in five fields randomly
selected from a section by measuring picrosirius red-stained
fibrosis area and dividing by the total myocardium area (four
or five sections of each left ventricle). Perivascular fibrosis was
estimated as the ratio of collagen-stained fibrosis surrounding
the vessel wall to the total vessel area (15–20 coronary arteries
for each animal). Areas of reparative fibrosis were not
considered in the analysis.
Group means7s.e. were calculated for all relevant variables.
Statistical comparisons were performed by ANOVA followed
by the Neumann–Keuls test. The limit of statistical signifi-
cance was set at Po0.05.
To investigate the effect of cariporide on pressure overload-
induced myocardial responses, mice were subjected to aortic
banding. Experimental measurements of cardiac hypertrophy
and function were taken at earlier (2 weeks) and later (5 weeks)
time points following surgery. To make sure that the effects of
cariporide were not mediated through indirect hemodynamics
effects, we measured the pressure gradient between the right-
and left-carotid arteries and the LVSP in both treated and
untreated banded groups 2 weeks after surgery. The admin-
istration of cariporide did not significantly influence the
pressure gradient across the aortic banding (vehicle-treated
banded mice,Veh-Bd, 5073mmHg;
banded mice, Car-Bd, 4974mmHg). Also, the LVSP was
not different in the untreated banded group (15175mmHg) vs
the cariporide-treated banded group (14974mmHg).
Effect of cariporide on cardiac hypertrophy
After 2 weeks from surgery, the left ventricular weight (left
ventricular weight-to-body weight ratio, LVW/BW) of banded
mice was significantly increased (47%) compared to sham
controls (Table 1, Veh groups). In contrast, cariporide-treated
banded animals showed a much lower enhancement of
ventricular weight (15%) (Table 1, Car groups), indicating
that the inhibition of the cardiac NHE-1 can markedly
attenuate the development of LVH. There were no significant
Table 1 Body weight and LV hypertrophic parameters in sham and banded mice 2 and 5 weeks after aortic surgery
2 Weeks 5 Weeks
BW, body weight; LVW/BW, left ventricular weight-to-body weight ratio; CSA, cardiomyocyte cross-sectional area. Veh-Sh: vehicle-
treated, sham-operated; Veh-Bd: vehicle-treated, banded; Car-Sh: cariporide-treated; Car-Bd: cariporide-treated, banded.
n¼number of experimental points; values are expressed as mean7s.e., *Po0.05 vs sham-operated groups;wPo0.05 vs all other groups.
British Journal of Pharmacology vol 141 (3)
G. Marano et al
LV function and myocardial NHE-1 inhibition
differences in LVW/BW ratio between cariporide-treated (Car-
Sh) and vehicle-treated sham mice (Veh-Sh), indicating that
the drug has no intrinsic effects on heart weight in the absence
of pressure overload (Table 1). Likewise, there were no effects
of cariporide on the overall BW gain (Table 1).
To analyze how this antihypertrophic effect of cariporide is
reflected by a change in the myocardium structure, the hearts
of both treated and untreated animals were subjected to
histological evaluation. Morphometric analysis of cross-
sections of left ventricular tissue revealed a significant increase
(49%) in cardiomyocyte cross-sectional area (CSA) in the
hearts of banded animals compared to controls, but this
increase was significantly smaller (19%) under cariporide
treatment (Table 1). Cariporide thus appears to reduce the
enhancement of the mean cellular area that accounts for the
early hypertrophic response. Again, no significant differences
were observed between sham mice groups (Table 1). In all
groups, there were minimal and nonsignificant changes in
collagen ventricular deposition and perivascular fibrosis 2
weeks after surgery (data not shown).
After 5 weeks from surgery, the enhancement of ventricular
weight induced by aortic banding was larger, both in
nontreated (65%) and cariporide-treated (30%) animals, but
the inhibitory effect of the antiporter blocker remained clearly
evident, and to an extension similar to that observed at 2 weeks
Morphometric analysis at 5 weeks confirmed a significant
decrease in cardiomyocyte hypertrophic response in Car-Bd
hearts compared with Veh-Bd ones (CSA, Table 1). However,
it also revealed an increased perivascular and interstitial
collagen deposition in both banded groups (Figure 1, a–c).
There was no difference in the intensity of collagen staining
between cariporide treatment and controls, suggesting that
cariporide has no influence on LV collagen deposition under
pressure overload conditions.
Effects of cariporide on LV remodeling and cardiac
To measure how cariporide affected ventricular performance,
echocardiography and cardiac catheterization were performed
both after 2 and 5 weeks from aortic banding surgery.
Following 2 weeks of chronic pressure overload, the
enlargement of cardiac mass in Veh-Bd was primarily
accounted for by concentric hypertrophy. In fact, the relative
wall thickness (RWT, calculated as ratio between posterior-
wall end-diastolic thickness and end-diastolic diameter) was
significantly increased (Table 2, Veh-Bd group), whereas the
systolic wall stress (ss) remained normal (Veh-Sh, 7675gcm–2;
Veh-Bd, 7877gcm–2), presumably because of the adequate
increase in the RWT.
In contrast, cariporide-treated banded animals displayed a
larger ss value (Car-Bd, 9476gcm–2), likely reflecting the
attenuated increase of wall thickness exerted by the drug
treatment. Remarkably, despite the blunting of the hyper-
trophic response and the resultant increase in the systolic wall
stress, cariporide-treated banded animals exhibited preserved
LV contractility and systolic function. These parameters were
weeks after surgery. Bar graphs show myocardial perivascular (a) and interstitial (b) fibrosis in left ventricles. Augmented
perivascular and interstitial fibrosis is apparent in banded groups (Veh-Bd and Car-Bd). Representative photomicrographs of cross-
sections of coronary arteries are shown (c). Picrosirius red-stained sections (?200) outline collagen (arrows). Veh-Sh, vehicle-
treated, sham-operated; Veh-Bd, vehicle-treated, banded; Car-Sh, cariporide-treated, sham-operated; Car-Bd, cariporide-treated,
banded. *Po0.05 vs sham groups.
Effect of banding with and without cariporide treatment on perivascular and interstitial myocardial collagen in mice 5
G. Marano et al
LV function and myocardial NHE-1 inhibition529
British Journal of Pharmacology vol 141 (3)
evaluated as Ees, a load-independent index of myocardial
contractility, and left ventricular fractional shortening (LV
FS), respectively (Table 2). Both were identical in cariporide-
treated and untreated sham animals. Moreover, as also shown
in Table 2, the maintained ventricular performance in
cariporide-treated banded animals cannot be explained by a
possible effect of the drug on preload (left ventricular end-
diastolic pressure, LVEDP), afterload (LVSP), or heart rate
(HR), that is, by indirectly affecting the hemodynamic loading.
Also, diastolic function (evaluated as LVEDP) did not
significantly differ among experimental groups (Table 2).
After 5 weeks of surgery, signs of cardiac dysfunction
induced by pressure overload became evident. Animals not
treated with cariporide (Veh-Bd in Table 2) exhibited a 16%
increase in ventricular end-diastolic dimension (LVEDD),
36% decrease in myocardial contractility (Ees), and a 26%
reduction in percent fractional shortening (%FS), as shown in
Table 2. These changes were accompanied by a 100% increase
in LVEDP compared with the sham control group (Table 2).
Cariporide appeared quite effective in contrasting such a
decline in heart performance, since echocardiograms revealed
the preservation of systolic function evaluated as %FS in the
Car-Bd (compare Veh-Bd and Car-Bd groups in Table 2). This
is also shown by ESPVRs recorded in Car-Bd. The slopes (Ees)
computed from such relationships were significantly steeper in
the Car-Bd than in the Veh-Bd (Table 2), which indicate intact
LV contractility. Representative ESPVRs recorded at 5 weeks
are presented in Figure 2.
In the present study, we demonstrate that cariporide treatment
has two major effects on the hearts of mice subjected to
pressure overload. One effect is inhibition of the hypertrophic
response, which is explained by a reduced enlargement of
myocardiocyte size. This is in agreement with previously
documented effects in in vitro as well as in vivo studies
(Yamazaki et al., 1998; Camilion de Hurtado et al., 2002;
Engelhardt et al., 2002), and extend to the mouse model of
aortic banding-induced LVH. In the mouse, the inhibitory
effect was present both at early and delayed stages after
surgery and was of similar magnitude at these time points. This
suggests that cariporide treatment may slow down the rate of
hypertrophy development rather than block the maximal
extent of the phenomenon.
The second effect is that cariporide can prevent (or delay)
the pathological remodeling and the deterioration of cardiac
systolic function at least up to 5 weeks of pressure overload.
This protective influence on cardiac function occurs despite the
lack of effect of cariporide on the cardiac fibrosis, observable
only after 5 weeks of surgery.
The antihypertrophic effect of cariporide is intriguing. In
fact, despite the marked blunting of the adaptive cardiac
hypertrophic response to mechanical overload, Car-Bd have a
systolic function as effective as that of control mice in which a
much larger LV mass is developed. These findings suggest at
least two possible explanations: (i) one is that in the earlier
stages, there is no proportionality between the degree of
hypertrophic response and extent of compensation. In other
words, the left ventricle may have the potential to cope with an
increased afterload even without becoming hypertrophic.
Similar findings were reported for other inhibitors of cardiac
hypertrophy, such as propranolol (Marano et al., 2002),
cyclosporin (Hill et al., 2000), or were inferred from the study
of mice deficient in catecholamine synthesis (Esposito et al.,
2002). However, controversy exists as to whether the abolition
of hypertrophy has detrimental effects on cardiac function.
Some studies (Meguro et al., 1999; Oie et al., 2000) found that
pharmacological inhibition of the LVH response induces a
more rapid decline in LV systolic function and progression to
failure, whereas according to other authors (Hill et al., 2000;
Esposito et al., 2002), the abolition of LVH is associated with a
preserved systolic function. Thus the functional implications of
pressure overload-induced LVH are still a matter of debate. (ii)
Another explanation is that inhibition of NHE-1 may enhance
myocardiocyte performance so that a smaller degree of
enlargement is sufficient to achieve the same extent of
compensation. The exact mechanism by which NHE-1
inhibition prevents pressure overload-induced cardiac hyper-
trophy and preserves LV systolic function under hemodynamic
overload conditions, remains to be elucidated. Changes in the
intracellular concentrations of sodium ion might be involved.
Sodium has been shown to exert hypertrophic effects in
cultured cardiac myocytes and a high Naþdiet induces cardiac
enlargement in hypertensive and normotensive rats (Frohlich
Invasive hemodynamics and echocardiographic parameters in sham and banded mice 2 and 5 weeks after aortic
2 Weeks5 Weeks
LV SP (mmHg)
LV FS (%)
HR, heart rate; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; LVEDD, left ventricular end-
diastolic diameter; RWT, posterior-wall thickness-to-left ventricular end-diastolic ratio; LV FS, left ventricular fractional shortening; Ees,
end-systolic elastance. Other abbreviations as in Table 1. n¼number of experimental points; values are expressed as mean7s.e., *Po0.05
vs sham-operated groups;wPo0.05 vs all other groups.
G. Marano et al
LV function and myocardial NHE-1 inhibition
British Journal of Pharmacology vol 141 (3)
et al., 1993; Gu et al., 1998). The amount of Naþentering
through the Naþ/Hþexchanger (NHE) accounts for approxi-
mately half the total Naþcellular influx and the intracellular
Naþwas found to be increased in pressure-overloaded hearts
(Frelin et al., 1984; Gray et al., 2001). Additional evidence
indicates that NHE-dependent sodium influx is a major
contributor to hypertrophy produced by various agonists,
including a1-adrenergic agonists, endothelin-1, or phorbol
esters, all of which act via the activation of various protein
kinase C (PKC) isoforms. NHE-1 inhibition attenuates both
the hypertrophy and the PKC activation (Hayasaki-Kajiwara
et al., 1999). An increase in intracellular sodium, via the
activation of cardiac Naþ/Ca2þ
intracellular calcium transients and the activation of calcium-
dependent signaling molecules such as PKC and transcription
factors. As a consequence, the beneficial effects of NHE-1
inhibition could be due to both changes in myocyte energy
handling and prevention of cytosolic Ca2þoverload through
reduction of Naþinflux.
The protective effect of cariporide on the derangement of
cardiac function induced by prolonged pressure overload is
important and may have important therapeutic implications.
However, also in this case the underlying mechanism is
unknown. We have shown in this study that cariporide
treatment contrasts the progression toward LV chamber
dilation and dysfunction. We also showed that cariporide
does not act by reducing arterial blood pressure, HR, or LV
filling, that is, its effect is independent of any attenuation in
cardiac mechanical overload. Again, this suggests that
cariporide can directly inhibit the cardiomyocyte remodeling
Our observations integrate and extend those from previous
studies, in which the beneficial effects of NHE-1 inhibition on
cardiomyocyte hypertrophy were observed in nonischemic
regions of the myocardium either in a postmyocardial
infarction model (Kusumoto et al., 2001), or in response to
enhanced b1-AR expression (Engelhardt et al., 2002). Thus
cariporide effects can be observed both in ischemic and
nonischemic tissues. However, attenuation of the increase in
myocyte size was not accompanied by a significant attenuation
of pressure overload-related interstitial fibrosis. This suggests
that at least in the earlier stages, the myocyte and the
nonmyocyte cellular components of myocardial tissue may
grow independent of each other and that the mechanisms
responsible for the growth of either component may be
different. In this regard, our data are in keeping with the
observations of Camilion de Hurtado et al. (2002) in the SHR
model, but are at variance with those from Engelhardt et al.
(2002) in the cardiac hypertrophic transgenic mouse with
overexpression of the b1-AR, in which cariporide was shown to
affect both cardiomyocyte hypertrophic and the interstitial
fibrotic processes. The discrepancy between their data and our
data may be related to the fact that different models of heart
failure were used. In our experimental model, LVH develops as
a response to mechanical overload rather than as a result of
genome manipulation as is the case for the b1-AR transgenic
mouse (Engelhardt et al., 2002). On the other hand, it is to be
considered that in the transgenic mice the treatment period was
longer than in our banded mice and that this might also be a
relevant factor considering that the regressions of myocyte
hypertrophy and collagen accumulation have different time
course (Cingolani et al., 2003).
The preserved cardiac function 5 weeks after aortic banding
could reflect a positive inotropic effect or hemodynamic effects
of cariporide. However, in cariporide-treated mice, the drug
affected neither cardiac contractility nor hemodynamic indices
such as preload, afterload, or HR. Taken together, these
findings suggest that the contribution of hemodynamic or
inotropic effects to the preserved cardiac function can be
excluded. In experimental models of cardiac hypertrophy and
exchanger, may reduce
sham (a), cariporide-treated banded (b), and vehicle-banded (c) mice
5 weeks after surgery. End-systolic pressure was plotted with end-
systolic volume to determine the end-systolic pressure–volume
relationship (ESPVR). Changes in hemodynamic load were obtained
by transitory caval occlusion (see text for details). Slope of the
regression line (ESPVR) is ventricular end-systolic elastance (Ees), a
load-independent index of cardiac contractility.
Representative pressure–volume relationships of vehicle-
G. Marano et al
LV function and myocardial NHE-1 inhibition 531
British Journal of Pharmacology vol 141 (3)
heart failure, cardiomyocyte apoptosis causes the progression
of compensated myocardial hypertrophy to LV dilation and
dysfunction (Condorelli et al., 1999). At present, we cannot
exclude the fact that the preserved cardiac function 5 weeks
after aortic banding was a result of reduced cardiomyocyte
In conclusion, we demonstrated in a mouse model of chronic
pressure overload that inhibiting the cardiac NHE-1 by
cariporide administration opposes the development of LVH
independent of any reduction in cardiac hemodynamic load,
and that this treatment favorably affects the progression to LV
pathological remodeling and systolic dysfunction.
This work was supported in part by the Ministry of Health Grants ICS
030.6/RF00-49 and 1AH/FW. We thank Aventis Pharma (Frankfurt,
Germany) for supplying the experimental diet.
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(Received October 22, 2003
Accepted November 10, 2003)
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LV function and myocardial NHE-1 inhibition
British Journal of Pharmacology vol 141 (3)