Ouabain-induced hypertension enhances left ventricular contractility in rats.

Abstract

Chronic ouabain treatment produces hypertension acting on the central nervous system and at vascular levels. However, cardiac effects in this model of hypertension are still poorly understood. Hence, the effects of hypertension induced by chronic ouabain administration ( approximately 8 microg day(-1), s.c.) for 5 weeks on the cardiac function were studied in Wistar rats. Ouabain induces hypertension but not myocardial hypertrophy. Awake ouabain-treated rats present an increment of the left ventricular systolic pressure and of the maximum positive and negative dP/dt. Isolated papillary muscles from ouabain-treated rats present an increment in isometric force, and this effect was present even when inotropic interventions (external Ca(2+) increment and increased heart rate) were performed. However, the sarcoplasmic reticulum activity and the SERCA-2 protein expression did not change. On the other hand, the activity of myosin ATPase increased without changes in myosin heavy chain protein expression. In addition, the expression of alpha(1) and alpha(2) isoforms of Na(+), K(+)-ATPase also increased in the left ventricle from ouabain-hypertensive rats. The present results showed positive inotropic and lusitropic effects in hearts from awake ouabain-treated rats, which are associated with an increment of the isometric force development and of the activity of myosin ATPase and expression of catalytic subunits of the Na(+), K(+)-ATPase.

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Ouabain-induced hypertension enhances left ventricular contractility in rats
Luciana V. Rossonia,⁎, Fabiano E. Xavierb, Cleci M. Moreirab, Diego Falcochiob,
Angélica M. Amansoa, Carolina U. Tanouea, Carla R.O. Carvalhoa, Dalton V. Vassallob,c
aDepartment of Physiological and Biophysical Sciences, ICB, University of Sao Paulo, Sao Paulo, SP, Brazil
bDepartment of Physiological Sciences, Federal University of Espirito Santo, Brazil
cDepartment of Physiological Sciences, EMESCAM, ES, Brazil
Received 23 January 2006; accepted 26 April 2006
Abstract
Chronic ouabain treatment produces hypertension acting on the central nervous system and at vascular levels. However, cardiac effects in this
model of hypertension are still poorly understood. Hence, the effects of hypertension induced by chronic ouabain administration (∼8 μg day−1, s.c.)
for 5 weeks on the cardiac function were studied in Wistar rats. Ouabain induces hypertension but not myocardial hypertrophy. Awake ouabain-
treated rats present an increment of the left ventricular systolic pressure and of the maximum positive and negative dP/dt. Isolated papillary muscles
from ouabain-treated rats present an increment in isometric force, and this effect was present even when inotropic interventions (external Ca2+
increment and increased heart rate) were performed. However, the sarcoplasmic reticulum activity and the SERCA-2 protein expression did not
change. On the other hand, the activity of myosin ATPase increased without changes in myosin heavy chain protein expression. In addition, the
expression of α1and α2isoforms of Na+, K+-ATPase also increased in the left ventricle from ouabain-hypertensive rats. The present results showed
positive inotropic and lusitropic effects in hearts from awake ouabain-treated rats, which are associated with an increment of the isometric force
development and of the activity of myosin ATPase and expression of catalytic subunits of the Na+, K+-ATPase.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Ouabain; Hypertension; Papillary muscle; Na+; K+-ATPase; SERCA-2; Myosin ATPase
Introduction
Digitalis has been used in clinical practice for over
200 years, since Willian Withering's original description in
1978, for their positive inotropic effect. The mechanisms of
action of digitalis have been under extensive investigation
for nearly 50 years, yielding one of the most specific
mechanisms thus far defined for any agent so extensively
used. Recent observations suggest that digitalis may have
additional effects on cardiac cell function in both the short
and long term (Wassertrom and Aistrup, 2004) and in
addition, can be fully accepted that ouabain, a digitalis
compound, is an endogenous regulator of blood pressure
and Na+, K+-ATPase activity (Hamlyn et al., 1996; Schoner
and Scheiner-Bobis, 2005).
Chronic ouabain treatment produces hypertension (Huang
et al., 1994; Manunta et al., 1994; Kimura et al., 2000;
Rossoni et al., 2002; Xavier et al., 2004). This hypertension
seems to be depending, at least in part, to an activation of
the central nervous mechanisms associated with increased
sympathetic tone, subsequent to the activation of the brain
renin–angiotensin (Huang et al., 1994; Huang and Leenen,
1999) and endothelin (Di Filippo et al., 2003) systems. In
addition, peripheral vascular mechanisms also contribute for
the maintenance of hypertension in this model (Kimura et
al., 2000; Rossoni et al., 2002; Di Filippo et al., 2003;
Xavier et al., 2004; Briones et al., 2006). On the other
hand, it is known that hypertension is associated with
structural, functional and biochemical adjustments on the
cardiac tissue (Swynghedauw, 1999). However, whether
hypertension induced by ouabain is associated or not with
Life Sciences 79 (2006) 1537–1545
www.elsevier.com/locate/lifescie
⁎Corresponding author. Departamento de Fisiologia e Biofísica, Instituto de
Ciências Biomédicas I, Universidade de São Paulo, Av. Professor Lineu Prestes,
1524, sala 101B, 05508-900 São Paulo, SP, Brazil. Fax: +55 11 3091 7285.
E-mail address: lrossoni@icb.usp.br (L.V. Rossoni).
0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2006.04.017

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changes of the heart function is not completely elucidated
yet.
Reports have been shown that models of hypertension
commonly produce a concentric cardiac hypertrophy by a
pressure overload mechanism. Although resulting from several
mechanisms it usually causes myocyte enlargement, collagen
accumulation, changes in the activity and expression of myosin
ATPase, shift of V1 to V3 myosin heavy chain (MHC) isoforms
and changes in Ca2+handling by reducing sarcoplasmic
reticulum activity (Mercadier et al., 1981; Tanamura et al.,
1993; Arai et al., 1996; Mill et al., 1998; Shorofsky et al., 1999;
Swynghedauw, 1999). In consequence, the alterations in the
mechanical properties of the myocardium together with the
prolongation of contraction duration and depressed shortening
velocity usually impair force development (Swynghedauw,
1999; Shorofsky et al., 1999).
Knowing the beneficial cardiac effects induced by digitalis
on the clinical practice, in the present moment it is extremely
important to acknowledge if the endogenous ouabain system is
able to affect the heart function. Studies that investigated
cardiac changes on long-term ouabain treatment were per-
formed only during 2 or 4 days in isolated adult cardiomyocytes
(Müller-Ehmsen et al., 2003) and papillary muscles (El-
Armouche et al., 2004). Moreover, the increment in blood
pressure induced by ouabain treatment does not develop within
this time.
Thus, the aim of the present study was to test, in addition to
the central nervous system and peripheral vascular mechan-
isms already described, if alterations in cardiac function might
occur as an adaptive response to the ouabain-induced
hypertension. In particular, we have investigated: (1) arterial
and left ventricular pressures in conscious rats, (2) ventricular
weights, (3) left ventricular papillary muscle contractility, (4)
activity and protein expression of myosin ATPase, (5) protein
expression of the Sarcoplasmic Endoplasmic Reticulum
Calcium-ATPase (SERCA) and (6) α1 and α2 isoforms of
the Na+, K+-ATPase. To our knowledge, this is the first time
that these parameters have been investigated in hearts from this
model of hypertension.
Material and methods
Animals
Six-week-old male Wistar rats were used in this study.
During treatment, rats were housed at a constant room
temperature, humidity and light cycles (12-h light/dark) had
free access to tap water and were fed with standard rat chow ad
libitum. Care and use of laboratory animals and all experiments
were conducted in compliance with the guidelines for
biomedical research as stated by the Brazilian Societies of
Experimental Biology.
Pellet implantation
Under anesthesia with diethyl ether, a small incision was
made on the back of the neck and one controlled time-release
pellet (Innovative Research of America, Florida, USA) contain-
ing either ouabain (0.5 mg pellet−1) or vehicle (placebo) was
implanted subcutaneously, as described (Rossoni et al., 2002).
These pellets are designed to release a constant amount of either
ouabain (≈8.0 μg/day) or vehicle for a 60-day period. The
length of treatment was 5 weeks. After 5 weeks of treatment the
following protocols were performed.
Arterial blood pressure and left ventricular pressure
measurements
Rats were anesthetized with ketamine/xylazine/aceproma-
zine (64.9; 3.20 and 0.78 mg/kg, i.p.) and allowed to breathe
room air spontaneously. A polyethylene catheter (PE50, 8 cm,
filled with heparinized saline) was introduced through the right
carotid artery into the left ventricle. During this procedure
arterial blood pressure and left ventricle systolic (LVSP) and
end diastolic pressure (LVEDP) and their first time derivatives
(positive and negative, dP/dtmax and dP/dtmin, respectively)
were recorded. When the polyethylene catheter was introduced
into the left ventricle it was fixed and the external part of the
catheter was exteriorized in the back of the neck through the
subcutaneous tissue. After 24 hLVSP and LVEDP and their first
time derivatives were recorded in awake animals. After that, the
LV catheter was pulled out and arterial blood pressure was
measured. The maintenance of diastolic blood pressure at
proper values was the guarantee that the aortic valve was not
damaged.
Arterial and ventricular pressures were recorded continu-
ously (Gould Statham P23XL transducer) in an 8-channel
recorder (Gould, model 5900). Heart rate and dP/dt were
determined by a biotech, triggered by pulse pressure and
recorded simultaneously with the other variables.
After this procedure rats received 500 units of heparin (i.p.)
and after 10 min they were anesthetized with 45 mg/kg of
sodium pentobarbital (i.p.), killed by exsanguination and then
the hearts were removed. For analysis of the activity and protein
expression of myosin ATPase and protein expression of
SERCA-2 and α isoforms of the Na+, K+-ATPase, hearts
were rapidly frozen in liquid nitrogen and kept at −80 °C until
the day of analysis.
Papillary muscles
The hearts were removed rapidly after thoracotomy and
perfused through the aortic stump to permit a proper selection
and dissection of the left ventricle papillary muscles. The
preparations were mounted in a plexiglass chamber continu-
ously superfused with gassed (95% O2and 5% CO2) Krebs
bicarbonate buffer solution, at 29±1 °C. Muscles stretched to
Lmax(muscle length at which active tension is maximal) were
stimulated by isolated rectangular pulses (10 to 15 V, 12 ms
duration) through a pair of platinum electrodes placed along the
entire extension of the muscle. The standard stimulation rate
was 0.5 Hz (steady state). Recording started after 60 min to
permit the beating preparation to adapt to the new environmen-
tal conditions.
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The bathing solution was a modified Krebs solution with the
following composition (in mM): 120 NaCl, 5.4 KCl, 1.2 MgCl2
6H2O, 1.25 CaCl22H2O, 2.0 NaH2PO4H2O, 1.2 Na2SO4, 27
NaHCO3, 11 glucose. Developed force (F) was measured with
an isometric force transducer (Nihon-Kohden, TB 612T, Tokyo)
and recorded on a chart recorder (Nihon-Kohden, RM-6200,
Tokyo), and normalized to the muscle cross-sectional area (g/
mm2). Considering the papillary muscle as a cylinder and tissue
density as 1, the cross-sectional area was calculated by dividing
the muscle length at Lmax by its weight (cross-sectional
area=0.99±0.045 and 1.00±0.062 g/mm2for vehicle and
ouabain-treated rats, respectively, P>0.05, t-test). To avoid the
possibility of a hypoxic core we performed experiments at low
temperature (29±1 °C).
The following protocols were used:
1. The effects of chronic ouabain treatment on the isometric
force development were compared to vehicle-treated rats
under control conditions and during changes in the rate of
stimulation (0.1, 0.25, 0.5, 0.75 and 1 Hz) using the protocol
previously described.
2. Under steady-state conditions force was measured at
different Ca2+concentrations (0.62; 1.25 and 2.5 mM) in
preparations from vehicle and ouabain-treated rats.
3. Post-rest potentiation was used to provide information
about the function of the sarcoplasmic reticulum (SR). The
force developed during the contraction of the cardiac
muscle is altered in response to changes in rate and rhythm.
In cardiac muscle, the contractions occurring after short
pauses are potentiated. In the rat cardiac muscle post-rest
contractions increase its force as the rest period increases
(Mill et al., 1992). Post rest contractions depend on pause
duration and on the amount of calcium stored at
intracellular sites and the relative participation of the SR
are more important for post rest contractions than for steady
state contractions. Pause intervals of various durations (15,
30 and 60 s) were used and the results are presented as
relative potentiation (the amplitude of post-rest contractions
divided by steady-state contractions) to normalize the data
from different preparations.
Measurements of activity of myosin ATPase
To evaluate if chronic ouabain treatment affected myosin
ATPase the enzyme activity was assayed as previously reported
(Moreira et al., 2003). Myosin was prepared from minced and
homogenized left ventricles, extracted briefly with KCl
phosphate buffer (0.3 M KCl, 0.2 M phosphate buffer, pH
6.5). After precipitation of myosin and muscle residues by 15-
fold dilution with water, the muscle residue was separated by
filtration using cheesecloth. This procedure filters fragments of
cells including membranes. The supernatant containing the
myosin was centrifuged at 33,000×g for 30 min. After
decantation of the supernatant the precipitate was redissolved
in 0.6 M KCl to elute myosin under high ionic strength and 1 ml
of water was added for each gram of tissue to produce a new
precipitation. The material was again centrifuged at 33,000×g
for 30 min and the muscle residue was separated by filtration.
The material was redissolved again in 14 ml of water per gram
of tissue, centrifuged, and filtered as before. The precipitate was
dissolved in 50 mM HEPES, pH 7.0, and 0.6 M KCl plus 50%,
v/v, glycerol and placed at −20 °C. To use the stocked myosin it
was diluted in water (1:12) and centrifuged at 3000 rpm for
15 min. The precipitate was resuspended in 50 mM HEPES, pH
7.0, and 0.6 M KCl, and centrifuged at 3000 rpm again. The
supernatant was used.
Myosin ATPase activity was assayed by measuring Pi
liberation from 1 mM ATP in the presence of 50 mM HEPES,
pH 7.0, 0.6 M KCl, 5 mM CaCl2, or 10 mM EGTA in a final
volume of 200 μl. Under this high ionic strength and no Mg2+in
the medium, only myosin activity was measured and there is no
significant Ca2+-ATPase activity from sarcoplasmic reticulum
membranes, which request high Mg2+and low Ca2+concentra-
tions. The nucleotide was added to the reaction mixture and
preincubated for 5 min at 30 °C. The reaction was initiated by
adding the enzyme fraction (3 to 5 μg protein) to the reaction
mixture. Incubation times and protein concentration were
chosen in order to ensure the linearity of the reaction. The
reaction was stopped by the addition of 200 μl of 10%
trichloroacetic acid. Controls with addition of the enzyme
preparation after addition of trichloroacetic acid were used to
correct for nonenzymatic hydrolysis of the substrate. All
samples were in duplicate. The enzyme activity was calculated
as the difference between the activities observed in the presence
of Ca2+and in the presence of 10 mM EGTA. The specific
activity was reported as nmol Pi released per minute per
milligram of protein unless otherwise stated. The total protein
content was measured using the method described by Bradford
(1976).
Protein expression of α and β myosin heavy chain isoforms
The myosin fraction was homogenized in 1 ml of water and
centrifuged at 10,000×g for 40 min. The pellet was resuspended
in Tris–EDTA buffer (pH 8.0) and the protein concentration
was measured using the method described by Bradford (1976).
Afterwards, the homogenates were boiled in 1.5 M Tris (pH
7.0), 0.25% SDS, 6.0% glycerol, 0.01% 2-mecaptoethanol and
0.0015% bromophenol blue buffer for 10 min and samples
(0.75 μg protein per lane) were electrophoretically separated on
a 7.5% SDS-PAGE. The gels were stained with 0.03%
Coomassie Blue stain and the signals on the blots were
densitometrically analyzed using the Scion Image software.
Protein expression of SERCA
Left ventricles were homogenized in 3 ml extraction buffer
(100 mM Trisma, pH 7.5; 10 mM EDTA; 10% sodium dodecyl
sulfate (SDS); 100 mM NaF; 10 mM sodium pyrophosphate;
10 mM sodium orthovanadate; at 100 °C) for 30 s. Samples
were boiled for 5 min, centrifuged and aliquots of supernatants
were used for the measurement of total protein content, as
described (Bradford, 1976). Proteins (100 μg) of each sample
were separated using 6.5% SDS-PAGE. The proteins in the gel
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were transferred to a nitrocellulose membrane. In the sequence
the membranes were incubated with anti-Serca-2 antibody
(Santa Cruz Biotechnology; Santa Cruz, CA, USA) at room
temperature, for 4 h. Membranes were washed and incubated
with anti-IgG antibody linked to horseradish peroxidase at room
temperature, for 1 h. Then membranes were incubated with
substrate for peroxidase and chemiluminescence enhancer
(Amersham Pharmacia Biotech) for 1 min and immediately
exposed to X-ray film for 1–10 min. Films were then revealed
in the conventional manner.
Protein expression of α1and α2isoforms of Na+, K+-ATPase
Protein expression of α1 and α2 isoforms of Na+, K+-
ATPase was measured using the method described by
Rossoni et al. (2002). Left ventricles were homogenized in
ice-cold sucrose–Tris–EDTA buffer (in mM: Tris—50,
Sucrose—250, EDTA—1.0, pH 7.4). Rat kidney microsomal
fractions were used as controls for the α1 and brain
microsomal fractions for the α2 isoforms. To prepare the
microsomal fractions of left ventricle, kidney and brain an
initial centrifugation was made at 10,000×g for 10 min at
4 °C. The supernatant was centrifuged at 100,000×g for
60 min. The pellet, representing the microsomal fraction,
was resuspended in Tris–EDTA buffer (in mM: Tris—50,
EDTA—1.0, pH 7.4) and the protein concentration was
measured as described by Bradford (1976). 45 μg protein
for left ventricle of ouabain-treated and vehicle rats, as well
as the corresponding controls (10 μg of protein each for
kidney and brain homogenates per lane) and prestained
molecular SDS-PAGE standards (Bio-Rad, Laboratories,
Hercules, CA, U.S.A.) were electrophoretically separated
on a 7.5% SDS-PAGE and then transferred to polyvinyl
difluoride membranes for 2 h at 4 °C, using a Mini Trans-
Blot Transfer Cell system (Bio-Rad) containing (in mM):
Tris—25, glycine—250, methanol—20% and SDS—0.05%.
Then the membrane was blocked for 60 min at room
temperature in Tris-buffered solution (in mM: Tris—25,
NaCl—137, Tween 20—0.2%, pH 7.5) with 5% powdered
non-fat milk. Next, the membrane was incubated overnight
at 4 °C with anti-α1 rabbit polyclonal IgG (0.1 mg ml−1
dilution) or anti-α2 rabbit polyclonal antiserum (1:5000
dilution), all purchased from Upstate Biotechnology (Lake
Placid, NY, U.S.A.). After washing, the membrane was
incubated for 90 min with an anti-rabbit IgG antibody
conjugated to horseradish peroxidase (1:3000 dilution; Bio-
Rad). The membrane was thoroughly washed and the
immunocomplexes were detected using an enhanced horse-
radish peroxidase/luminol chemiluminescence system (ECL
Plus, Amersham International plc, Little Chalfont, U.K.) and
then subjected to autoradiography for either 5 min (α1) or
10 min (α2).
Drugs used
Pentobarbital sodium (Cristalia—Produtos Químicos Farm-
acêuticos Ltd., São Paulo, SP, Brazil); Heparin (Roche Q.F.S.
A., Rio de Janeiro, RJ, Brazil; Sigma) and Caffeine anhydrous
were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.
A.). All other reagents used were of analytical grade from
Sigma; E. Merk (Darmstadt, Germany) or Reagen (Rio de
Janeiro, RJ, Brazil).
Statistical analysis
The results are presented as mean±SEM with N indicating
the number of observations. Values were analyzed using t-test or
ANOVA (one- and two-way). When ANOVA revealed a
significant difference, Tukey test was applied. P<0.05 was
taken as significant. The analysis of the data and the plotting of
figures were carried out using softwares GraphPad Prism™
(version 3.0, GraphPad Software, San Diego, CA, USA) and
GB-STAT (version 4.0, Dynamic Microsystem Inc., Silver
Spring, MD).
Results
Arterial blood pressure and left ventricular pressure in awake
animals
Five weeks of ouabain treatment did not affect the body
weight of the rats (ouabain: 264±4.17 vs. vehicle: 283±11.9 g;
P>0.05) and no signs of left (ouabain: 2.18±0.05 vs. vehicle:
2.14±0.07 mg/g; P>0.05) or right (ouabain: 0.62±0.03 vs.
vehicle: 0.58±0.02 mg/g; P>0.05) ventricular hypertrophy
were observed.
As previously reported, in conscious ouabain-hypertensive
rats systolic and diastolic arterial pressures increased compared
to the vehicle group and no differences were observed in heart
rate (Table 1). However, novel hemodynamic results observed
in the present study are the increment of LVSP, dP/dtmaxand dP/
dtminin conscious ouabain-hypertensive compared to vehicle
rats and no changes on LVEDP (Table 1).
Isolated papillary muscles
Fig. 1A shows that in control conditions (rate of stimulation
of 0.5 Hz and 1.25 mM CaCl2) papillary muscles from ouabain-
hypertensive rats developed larger forces compared to the
Table 1
Changes in systolic (SBP) and diastolic blood pressure (DBP), in heart rate
(HR), in left ventricle systolic pressure (LVSP) and end diastolic pressure
(LVEDP)andinpositive(dP/dtmax)andnegativefirst time derivatives(dP/dtmin)
obtained from conscious vehicle (n=10) and ouabain-treated (n=10) rats
Vehicle Ouabain
146±3.16⁎
97±3.35⁎
354±8.88
142±0.84⁎
7.20±0.79
7760±167⁎
−7016±157⁎
SBP (mm Hg)
DBP (mm Hg)
HR (bpm)
LVSP (mm Hg)
LVEDP (mm Hg)
dP/dtmax(mm Hg/s)
dP/dtmin(mm Hg/s)
t-Test, P<0.05,⁎vs. vehicle.
126±2.11
88±2.31
341±8.99
123±1.11
7.83±1.45
6617±189
−6105±212
1540L.V. Rossoni et al. / Life Sciences 79 (2006) 1537–1545

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vehicle group. To investigate if ouabain treatment affected the
action of inotropic interventions, changes in heart rate and
concentration-response curve to CaCl2 were performed. As
expected for the rat myocardium the increase of the rate of
stimulation reduced force in both groups (Fig. 1B). Although
force was higher in the ouabain-treated animals for all rate
changes, the magnitude of force reduction was similar in both
groups (magnitude of force reduction is expressed as a
percentage of the response to 0.1 Hz taken this response as
the maximal force development for each papillary muscle:
Ouabain-treated: 57±2.99 vs. vehicle: 63±2.64% to 1 Hz rate
of stimulation, P>0.05) (Fig. 1B). Extracellular Ca2+increment
increased force in both groups, and, as observed before, the
ouabain-treated animals developed more force compared to
vehicle group (Fig. 1C).
Following that, chronic ouabain effects on the sarcoplasmic
reticulum were investigated. No differences in the relative
potentiation obtained in ouabain-hypertensive and vehicle-
0.0
2.5
5.0
7.5
Vehicle (9)
Ouabain (10)
*
A
Force (g/mm2)
0.620 1.2502.500
2
3
4
5
Vehicle (6)
Ouabain (7)
+
+
+
+
*
*
C
CaCl2 (mM)
Force (g/mm2)
0.00 0.25 0.500.75 1.001.25
0.0
2.5
5.0
7.5
Vehicle (7)
Ouabain (7)
+
+
+
+
+
+
*
B
Frequence (Hz)
Force (g/mm2)
Fig. 1. (A) Comparison of developed force in control conditions; (B) under changes in rate of stimulation and (C) increasing extracellular Ca2+concentration. Columns
or symbols represent mean±SEM. P<0.05,+vs. control conditions and⁎vs. vehicle.
0 1020 304050 60 70
1.0
1.1
1.2
1.3
1.4
Vehicle (8)
Ouabain (10)
A
Time (sec)
RP/ PRC
VehicleOuabain
0
50
100
150
200
B
AU (%)
125
101
SERCA 2
Vehicle Ouabain
Fig. 2. (A) Changes of relative potentiation after post rest contractions (RP/PRC) and (B) densitometric analysis of the Western blot of SERCA-2 protein expression
from left ventricle obtained from vehicle and ouabain-hypertensive rats. The inset in the (B) shows the representative Western blot for SERCA-2 protein expression of
left ventricle obtained from vehicle and ouabain-hypertensive rats. Western blot results are expressed as a percentage of the signal of SERCA-2 protein of the
corresponding left ventricle from vehicle-treated rats. Columns or symbols represent mean±SEM.
1541L.V. Rossoni et al. / Life Sciences 79 (2006) 1537–1545

Page 6

treated muscle were observed (Fig. 2A). In addition, after
5 weeks of ouabain treatment no difference was observed for
SERCA-2 protein expression when compared to the vehicle
group (Fig. 2B).
The possibility of actions of chronic ouabaintreatment on the
contraction machinery was also studied by measuring the
activity of myosin ATPase and the protein expression of myosin
heavy chain isoforms. The activity of myosin ATPase was
higher in the ouabain-hypertensive muscles (Fig. 3A). Howev-
er, protein expression of both α and β isoforms of myosin heavy
chain was similar in ouabain and vehicle groups (Fig. 3B).
As the main site of ouabain action is the catalytic subunits of
Na+, K+-ATPase we also measured the protein expression of α1
and α2isoforms of the Na+, K+-ATPase. Ouabain treatment
increased both α1and α2protein expression of Na+, K+-ATPase
in the left ventricle as compared to the vehicle group (Fig. 4).
Discussion
In this study we demonstrated for the first time that chronic
treatment with ouabain for 5 weeks, which induces hyperten-
sion, is associated with an increase of the ventricular inotropic
and lusitropic functions. This treatment did not produce cardiac
hypertrophy nor altered the function of the sarcoplasmic
reticulum but increased the activity of the myosin ATPase and
the protein expression of the catalytic α1and α2isoforms of the
Na+, K+-ATPase.
Similar to previous results (Huang et al., 1994; Manunta et
al., 1994; Kimura et al., 2000; Rossoni et al., 2002; Di Filippo et
al., 2003; Xavier et al., 2004; Briones et al., 2006) the present
work reinforce the hypothesis that chronic treatment with
ouabain in Wistar rats induces hypertension and did not change
heart rate. As described in the Introduction, the mechanisms
Vehicle
(7)
Ouabain
(7)
400
500
600
700
*
A
Myosin ATPase
(nmol Pi/min/mg)
Alpha-MHCBeta-MHC
0
25
50
75
100
B
Vehicle (5)
Ouabain (5)
MHC Isoforms
(% of total MHC)
Fig. 3. Comparisons of myosin ATPase activity (A) and protein expression of alpha- and beta-myosin heavy chain (MHC) isoforms (B) of the left ventricle from
vehicle and ouabain-hypertensive rats. Western blot results are expressed as a percentage of total myosin heavy chain (MHC) protein content present in the left
ventricle from vehicle or ouabain-treated rats. Columns represent mean±SEM. P<0.05,⁎vs. vehicle.
α α1Isoform
α α2Isoform
KidneyBrain
Ouabain-+
VehicleOuabain
0
250
500
750
*
α α1Isoform
AU (%)
Vehicle Ouabain
0
250
500
750
*
α α2 Isoform
AU (%)
Fig. 4. Upper panel—representative Western blots for α1and α2isoforms protein expression in intact segments from left ventricle of Wistar rats, which received
ouabain(+) or vehicle (−) subcutaneously for 5 weeks. The first and last line shows the correspondingpositive control for each protein (kidney for α1and brain for α2).
Lower panel—densitometric analysis of the Western blot of α1and α2protein expression in intact segments from left ventricle of Wistar rats that received ouabain
(N=4) or vehicle(N=4) for 5weeks.Resultsare expressedas a percentage of the signalof the α1or α2isoformsof the corresponding left ventriclefromvehicle-treated
rats. P<0.05,⁎vs. vehicle.
1542 L.V. Rossoni et al. / Life Sciences 79 (2006) 1537–1545

Page 7

involved on the genesis and/or maintenance of this model of
hypertension are associated with changes in the central nervous
system pathways (Huang et al., 1994; Huang and Leenen, 1999;
Di Filippo et al., 2003) and in the arterial function and
remodeling (Kimura et al., 2000; Rossoni et al., 2002; Di
Filippo et al., 2003; Xavier et al., 2004; Briones et al., 2006).
However, there are no reports describing changes in heart
function in the ouabain-hypertensive rats.
In conscious rats, we found suggestions that the contractile
activity was enhanced in ouabain-hypertensive animals because
an increment of the LVSP and of the positive dP/dtmaxwas
observed. In addition, this hypertensive treatment also increased
lusitropic parameters as observed by an increment in negative
dP/dt. Moreover, the isolated left ventricular papillary muscles
from ouabain-hypertensive rats developed more force when
compared to the vehicle group, which also could explain the
increment of the positive dP/dtmax reflecting an increased
inotropic state. This force increment is maintained even when
the muscle is under inotropic interventions like increased
extracellular Ca2+concentration or changes in the rate of
stimulation. This is an interesting finding that suggests that the
positive inotropic action of ouabain can be superimposed to
other positive inotropic actions without blunting them.
Reinforcing these results, El-Armouche et al. (2004) showed
in left ventricular papillary muscle from ouabain-treated rats for
4 days a sensitization for the positive inotropic effect of
isoprenaline.
In the rat myocardium both positive inotropic and lusitropic
effects are usually dependent on the activity of the sarcoplasmic
reticulum. Older reports suggested that ouabain reduces calcium
uptake (Lee and Choi, 1966; Carsten, 1967) and potentiates
calcium release from the sarcoplasmic reticulum (Fugino et al.,
1979; McGarry and Williams, 1993). More recently, Sagawa et
al. (2002) reported that cardiac glycosides amplify sarcoplasmic
reticulum calcium release by a luminal calcium sensitive
mechanism. Acting together those mechanisms contribute to
increase myoplasmic calcium concentration enhancing contrac-
tion. However, all these findings were obtained with acute
ouabain administration.
We then investigated possible changes of the sarcoplasmic
reticulum function, after chronic ouabain administration, using
the relative potentiation, obtained after pauses of increasing
duration performed in isolated left ventricular papillary
muscles (Mill et al., 1992), and by measuring the SERCA-2
protein expression. Our findings did not reveal changes in the
relative potentiation and these results were reinforced by the
fact that SERCA-2 protein expression did not change after
ouabain treatment. In agreement, Müller-Ehmsen et al. (2003)
using cultured rat cardiomyocytes treated for 2 or 4 days with
ouabain and El-Armouche et al. (2004) using the infusion of
this compound for 4 days in Wistar rats also showed that these
treatments did not change the transduction pathway involved
in calcium handling as the expression of SERCA-2a,
phospholamban, calsequestrin or ryanodine receptor. On the
other hand, in spontaneously hypertensive rats (SHR) the
larger force increment developed by papillary muscles, when
compared to controls, is abolished after treatment with
ryanodine (Mill et al., 1998). However, previous report
showed that the enhanced contractility found in the SHR
was not associated with any changes in the density of
ryanodine receptors, Ca2+-ATPase or phospholamban but to
the larger average amplitude of Ca2+sparks (Shorofsky et al.,
1999).
Hypertension produces cardiac overload and it is usually
associated with ventricular hypertrophy, and this hypertrophy is
associated with changes in the contractile machinery as changes
in myosin ATPase activity and in MHC isoforms (Mercadier et
al., 1981; Swynghedauw, 1999). Knowing that changes in
myosin ATPase activity can change the ventricular contractility
we performed protocols to investigate this activity. Indeed, we
observed for the first time an increment of myosin ATPase
activity on the left ventricular tissue from ouabain-hypertensive
rats. This fact could help to explain the enhanced contractile
activity of papillary muscles in the control condition. We also
investigated the isoforms of the myosin heavy chain but,
different from the myosin ATPase activity, the protein
expression of α and β isoforms did not change. The relationship
among myosin ATPase activity with larger percentage of α-
MHC and the isometric force development is a fact already
established (Barany, 1967; Swynghedauw, 1986, 1999; Tana-
mura et al., 1993). However, in the ouabain-treated hearts the
increments of myosin ATPase activity and of the isometric force
development are not associated to changes on the MHC
isoforms. Although existing this positive correlation between
the myosin ATPase activity and the abundance of α-MHC there
are other reports (Bartunek et al., 2000) showing that in L-
NAME-treated rats cardiac inotropism can be increased without
alteration of the isoform composition of the cardiac muscle, as
reported for the ouabain treatment model.
Additionally, the present study also demonstrated that
ouabain-induced hypertension occurs without cardiac hypertro-
phy, despite of increased left ventricular function and the
elevated arterial blood pressure. This result is very interesting,
although reports show that ouabain induces hypertrophy in
isolated cardiac myocytes by partial inhibition of the Na+, K+-
ATPase activity (Huang et al., 1997; Xie et al., 1999).
Previously, we demonstrated that ouabain-induced hypertension
is associated with regional changes in the activity of the
ouabain-sensitive sodium pump and of the expression of the α1
and α2Na+, K+-ATPase isoforms in the vascular smooth muscle
(Rossoni et al., 2002). Kent et al. (2004) also showed that
chronic treatment with ouabain for 14 days directly inhibits
Na+, K+-ATPase activity in the hypothalamus and up-regulated
the α3isoform expression while α1and α2expression remains
unchanged. Our findings reinforce those results, because in the
left ventricle from ouabain-hypertensive rats there is an
increment in the α1and α2isoforms of Na+, K+-ATPase. Gao
et al. (2002) showed that low concentration of digitalis
compounds activate the cardiac Na+, K+-ATPase activity
instead of inhibiting it. On the other hand, as described above,
it is known that the myocyte's hypertrophy and transcriptional
regulations of growth-related genes induced by in vitro ouabain
treatment are partially dependent of the Na+, K+-ATPase
inhibition (Huang et al., 1997; Xie et al., 1999). Supported by
1543 L.V. Rossoni et al. / Life Sciences 79 (2006) 1537–1545

Page 8

these results, we can speculated that, in vivo, cardiac
hypertrophy does not develop, as in cultured cardiac myocytes,
probably because of the activation of α1and α2isoforms of the
Na+, K+-ATPase by low doses of ouabain, as used in the present
study.
In the rat heart approximately 75% of the alpha Na+, K+-
ATPase protein isoform is α1; however, α2 and/or α3 are
consistently found in strategic sites (Blaustein et al., 1998). It is
know that the main site for ouabain action is assumed to be the
α-subunit of the Na+, K+-ATPase. The inhibition of the sodium
pump results in Na+accumulation in the myoplasm which
reduces the activity of the Na+/Ca2+exchange mechanism and
ultimately increases cardiac and vascular smooth muscle
contraction (Blaustein et al., 1998). If ouabain increases the
protein expression of α1and α2isoforms of Na+, K+-ATPase,
changes in the activity of the sodium pump could contribute to
the increment in the inotropic effect occurring in the ouabain-
hypertensive rats. In addition, recently it was demonstrated that
acute infusion of ouabain acting on α2isoforms of Na+, K+-
ATPase enhances the myocardial contractility in mice (Dostanic
et al., 2005). Our results also agree with Xie et al. (1999) and
Xie and Askari (2002), which suggest that it may be possible to
dissociate the positive inotropic effects induced by ouabain
from its cardiac hypertrophic effect.
Potential limitations of the study
In the present study we used fluid-filled manometric system
as a method to perform the hemodynamic experiments in
conscious animals. If we compared the present results with
results performed using microtip pressure transducers we
observed that the present values obtained with polyethylene
catheter are lower when compared to those obtained with the
microtip catheter (Capasso et al., 1992; Pacher et al., 2004;
Samsamshariat et al., 2005). However, the results using the
microtip catheter are commonly performed in anesthetized rats.
As the anesthetic state changes the hemodynamic parameters in
ouabain-hypertensive animals, we used the fluid-filled mano-
metric system to perform the present experiments knowing
about the resonance effect of this catheter and the dumping
produced by this manometric system. However, as the fluid-
filled manometric system was the same to perform all
experiments and, in addition, the results obtained on the
isolated left ventricle papillary muscle agree with the hemody-
namic data we believe that the differences obtained between the
groups in the present study are acceptable.
This is the first demonstration that hypertensive ouabain-
treated rats present positive left ventricular inotropism and
lusitropism. Our results suggest that the left ventricular
inotropic effect observed in papillary muscle and in awake
ouabain-hypertensive animals does not depend on changes of
the sarcoplasmic reticulum function or hypertrophy develop-
ment. Indeed, it is associated with an increased activity of the
myosin ATPase and an increased protein expression of catalytic
subunits of Na+, K+-ATPase. All these mechanisms are
superimposed by an increment in the sympathetic activity
(Huang et al., 1994; Huang and Leenen, 1999; Di Filippo et al.,
2003) that potentiates the positive inotropic and lusitropic
mechanisms in these hearts.
Acknowledgments
We thank Luciene M. Ribeiro for the technical assistance.
This study was supported by grants from CNPq, FAPESP and
CAPES.
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