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Vol. 298, Issue 2, 469-476, August 2001
Departments of Pharmacology and Toxicology (L.C., X.T.G., J.V.H., M.K.), Medicine (Q.F., X.L.), and Pathology (S.C.), University of Western Ontario, London, Ontario, Canada
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Pulmonary hypertension results in compensatory right ventricular (RV) hypertrophy. We studied the role of the Na+-H+ exchange (NHE) in the latter process by determining the effect of the NHE-1 inhibitor cariporide after monocrotaline-induced pulmonary artery injury. Sprague-Dawley rats received a control or cariporide diet for 7 days, at which time they were administered either monocrotaline (60 mg/kg) or its vehicle. Twenty-one days later, monocrotaline control, but not cariporide-fed animals, demonstrated increased RV weights and cell size of 65 and 52%, respectively. Monocrotaline alone significantly increased RV systolic pressure and end diastolic pressure by 70 and 94%, respectively, whereas corresponding values with cariporide were significantly reduced to 33 and 42%. Central venous pressure increased by 414% in control animals, which was significantly reduced by cariporide. Monocrotaline treatment produced a decrease in cardiac output of 28 and 8% in the absence or presence of cariporide (P < 0.05 between groups), respectively. Although body weights were significantly lower in both monocrotaline-treated groups compared with vehicle treatment, with cariporide the net gain in body weight was twice that seen in the monocrotaline-treated animals without cariporide. Monocrotaline also increased RV NHE-1 and atrial natriuretic peptide mRNA expression, which was abrogated by cariporide. Monocrotaline-induced myocardial necrosis, fibrosis, and mononuclear infiltration was completely prevented by cariporide. Cariporide had no effect on monocrotaline-induced pulmonary intimal wall thickening. Our results demonstrate that cariporide directly attenuates myocardial dysfunction after monocrotaline administration independent of pulmonary vascular effects. NHE-1 inhibition may represent an effective adjunctive therapy that selectively targets myocardial hypertrophic responses in pulmonary vascular injury.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Pulmonary
hypertension represents an important clinical problem that can result
in development of right ventricular (RV) hypertrophy and heart failure
(Krowka, 2000
; Gaine and Rubin, 1998) and which is associated
with a high rate of mortality (Lilienfeld and Rubin, 2000). The
ventricular hypertrophic response is probably mediated to a large
degree by the production of paracrine and autocrine hypertrophic agents
linked to signal transduction-dependent regulation of transcriptional
factors. Recent studies suggest that left ventricular myocardial
hypertrophy following infarction is dependent at least in part on the
activity of the sodium-hydrogen exchange (NHE), an electroneutral
antiporter representing the primary pathway for proton extrusion in the
cardiac cell in exchange for sodium ion influx (reviewed in Karmazyn et
al., 1999; Cingolani, 1999). Accordingly, inhibiting the primary NHE
isoform found in the myocardium (NHE-1) reduces both the hypertrophic
as well as heart failure responses after infarction in rats (Yoshida
and Karmazyn, 2000; Kusumoto et al., 2001). Further supporting the
concept of NHE-1 involvement are studies demonstrating reduced
hypertrophy in cultured cells exposed to various hypertrophic factors
when treated with NHE inhibitors (Hori et al., 1990; Yamazaki et al.,
1998).
Although emerging evidence supports NHE involvement in postinfarction
left ventricular failure, nothing is known about the role of the
antiporter in right ventricular hypertrophy secondary to pulmonary
hypertension. Among the most widely studied animal models of right
ventricular hypertrophy and right heart failure is the administration
of the pyrrolizidine alkaloid monocrotaline (MCT), which produces a
relatively rapid (within a few weeks) pulmonary hypertension resulting
in compensatory right ventricular hypertrophy and heart failure
(Rosenberg and Rabinovitch, 1998; Schultze and Roth, 1998;
Nakazawa et al., 1999; Pichardo et al., 1999). This experimental
approach is advantageous in the sense that the myocardial involvement
occurs secondarily to initiating factors that are of noncardiac origin,
thus allowing the possibility of closer mechanistic assessment of
therapeutic strategies. Moreover, as NHE inhibitors are also protective
agents in the ischemic myocardium, this approach also permits the
assessment of the effect of NHE-1 inhibition on myocardial hypertrophy
and heart failure independent of infarct size reduction. We therefore
hypothesized that NHE-1 inhibition could attenuate the right
ventricular hypertrophic response after MCT administration.
Accordingly, we examined the effect of the NHE-1-specific inhibitor
cariporide on the myocardial responses 3 weeks after MCT administration
to rats.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Experimental Protocol. One hundred and eight virus-free male Sprague-Dawley rats weighing 200 to 250 g were maintained in the Health Sciences Animal Care facilities of the University of Western Ontario in accordance with the guidelines of the Canadian Council on Animal Care (Ottawa, ON, Canada). The animals were randomly assigned to either the control chow or an identical diet containing 3000 parts per million of cariporide (Aventis Pharma, Frankfurt, Germany), which resulted in mean plasma levels of the drug of 381 ± 29 ng/ml (n = 10). After 1 week of feeding, the animals were randomized to receive subcutaneously administered MCT (60 mg/kg b.wt., Sigma-Aldrich Canada Ltd, Oakville, ON, Canada) or equal volumes of vehicle. MCT was prepared by dissolving it in 1 N HCl. The pH was neutralized with 0.5 N NaOH, and the volume of the solution was adjusted with phosphate-buffered saline (pH 7.40) to achieve a concentration of 30 mg/ml. Animals were subsequently maintained for a further 21 days on their respective diets, at which time experimental data were collected as described below. In addition, plasma was collected from 10 cariporide-treated rats (5 sham, 5 MCT-treated) for determination of plasma drug concentrations.
Hemodynamic Measurements. The animals were weighed and then anesthetized with sodium pentobarbital sodium (50 mg/kg b.wt., i.p.) and buprenorphine (0.03 mg/kg b.wt., s.c.). Animals were then intubated and ventilated (tidal volume, 10 ml/kg; ventilator rate, 70 breaths/min) with air using a rodent respirator (model 683, Harvard Apparatus, Holliston, MA). A standard lead II electrocardiogram was obtained using a Grass model 7P6 recorder (Astro-Med, West Warwick, RI), and a thermistor probe was inserted into the rectum to measure core temperature, which was maintained at 37-38°C with the aid of a heating pad. Arterial pressure and left ventricular pressure were sequentially measured with a PE-50 Intramedic polyethylene tube (0.023-inch i.d. and 0.038-inch o.d., Clay Adams, Parsippany, NJ) introduced into the right carotid artery by the neck cut-down and then advanced to the ascending aorta and the left ventricles, respectively. Central venous pressure and right ventricular pressure were sequentially measured with a PE-50 polyethylene tube that was slightly modified by heating and introduced into the right external jugular vein and advanced to the right atrium and the right ventricle, respectively. The catheters were connected to a pressure transducer (Model PX-1800, Baxter Healthcare Co., Irvine, CA) positioned at the mid-thorax of the animal. The transducers were connected to a BioPac Computer Data Acquisition System (Harvard Apparatus). The position of the catheter was confirmed by the waveform of the pressure tracing from the computer screen using AcqKnowledge software (version 3.2, Harvard Apparatus). After the catheterization procedures, a 10-min stabilization was allowed before the measurements were recorded and stored in the computer at a sampling rate of 100 Hz.
To assess cardiac output, the chest was opened by the middle sternal incision. The thymus and heart were exposed, and the right pleura was maintained intact. The thymus was then divided into the left and right lobe to expose the aorta, and the ascending aorta was gently separated from the pulmonary artery. A Transonic flowprobe connected to a T206 Transonic flowmeter (Transonic Systems Inc., Ithaca, NY) was placed around the ascending aorta. A chest tube was placed percutaneously, and the thoracotomy was closed in layers. The chest was then evacuated to allow inflation of the lung. A 10-min stabilization following surgery was allowed before the digital readings of cardiac output over a 1-min period were recorded. The animals were sacrificed at the end of the hemodynamic measurement by an overdose of pentobarbital administered intravenously via the right ventricular catheter. The heart and lungs were removed en bloc for further analysis (see below).Determination of Heart Weights. The heart was removed, the chambers were opened, and any excess blood was dried with paper towels, after which the heart was weighed. The atria and large blood vessels were then removed. The hearts were separated into the right ventricle free wall (RV) and the left ventricle (LV) including the septum and weighed separately.
Histology. Myocardial tissues (LV and RV, n = 10) were fixed by immersion in 10% buffered formalin and embedded in paraffin for morphological analyses. Five-micrometer sections stained with hematoxylin and eosin as well as Gomori's trichrome stain were used for morphological analyses. The samples were coded and the slides were subjected to light microscopic examination by one of the authors (S.C.), who was unaware of the specimen identity or treatment group.
NHE-1 and ANP mRNA Expression. Total RNA was isolated from either right or left ventricles and washed with 500 µl of ethanol (75% v/v) in diethyl pyrocarbonate (DEPC) water. The tubes were recentrifuged at 12,000g for 5 min, and the ethanol was decanted. All the RNA pellets were air-dried and redissolved in 50 to 100 µl of DEPC water. The RNA concentration was measured spectrophotometrically using an RNA/DNA calculator at 260 nm (Genequant II, Amersham Pharmacia Biotech Ltd., Cambridge, UK).
We determined NHE-1 expression using competitive reverse transcriptase polymerase chain reaction. Competitors were created using commercial DNA construction kits (Takara Shuzo, Otsu, Japan) such that the sequences at both ends of the competitor were complementary to the same primers for amplifying the target DNA and the sizes of the competitor DNA fragments were comparable with the target DNA fragments, assuring the same dynamics of the amplification reaction for the target and the competitor DNA fragments. Optimal competitor concentrations for each target gene were established by viewing the electrophoresis band pattern with serial dilutions of the competitor. The amplification was carried out using the NHE-1, primer 1 [(+), 5'-TCTGTGGACCTGGTGAATGA-3'] and primer 2 [(
),
5'-GTCACTGAGGCAGGGTTGTA-3'] with a predicted product size of 210 bp
and a competitor size of 292 bp. Reactions were performed in 25-µl
volumes containing 2.5 µl of 10× PCR-buffer, 1 µl of 50 mM
MgCl2, 1.25 µl of deoxynucleotide triphosphate
(dNTP) mix, 1.25 µl of each amplification primer, 0.25 µl of
Platinum Taq polymerase, 1 µl of the RT product and 1 µl
of the competitor in optimal concentration. The initial cycle was
carried out using 3 min at 94°C for denaturation, 1 min at 54°C for
annealing, and 3 min at 72°C for extension. Subsequent cycles of PCR
were performed using the following conditions: denaturation, 45 s
at 94°C; annealing, 45 s at 54°C; extension, 1 min at 72°C;
and final extension, 7 min at 72°C. The linearity of the PCR reaction
was established by analyzing the PCR product with variable amounts of
template and competitor and variable cycle numbers. In our study, we
used 30 cycles of amplification for NHE-1.
Semiquantitative determination of ANP mRNA was assessed by RT-PCR. For
reverse transcription of mRNA, 3 µg of total RNA from each sample
were used. Oligo(dT) (0.5 µg) and DEPC water was added to each PCR
tube to a final volume of 12 µl. The tubes were subsequently heated
at 70°C for 10 min exposing the poly(A) tails characteristic of mRNA
to the oligo(dT), thereby priming the mRNA for reverse transcription.
Next, dNTPs (0.5 mM), dithiothreitol (0.5 mM), Moloney murine leukemia
virus reverse transcriptase (100 units, Invitrogen,
Burlington, ON, Canada), and first strand buffer (1×, Invitrogen) were
added to a final volume of 20 µl to each PCR tube. Finally, the RNA
was reverse transcribed into cDNA by reverse transcriptase at 37°C
for 1 h.
Two microliters of cDNA from each sample was placed into PCR
reaction tubes (0.2-ml ultrathin wall). Subsequently, PCR buffer (1×,
Invitrogen), dNTPs (200 µM), MgCl2 (1.5 mM),
predesigned forward and reverse primers (1.5 µmol), Taq
polymerase (0.5 units, Invitrogen), and sterile
H2O to a final volume of 25 µl were added to
each PCR reaction tube. The forward and reverse primers for rat ANP
were 5'-CTGCTAGACCACCTGGAGGA-3' and 5'-AAGCTGTTGCAGCCTAGTCC-3', respectively. The forward and reverse primers for glyceraldehyde phosphate dehydrogenase were 5'-AAAGGGCATCCTGGGCTACA-3' and
5'-CAGTGTTGGGGGCTGAGTTG-3', respectively. The PCR tubes were then
placed in the thermal cycler, and a heat-cycling program was executed.
The melting temperature for the cDNA was set at 94°C, and the
Taq polymerase activation temperature was set at 72°C.
Annealing temperature for ANP and GAPDH was 60 and 66°C,
respectively. Equal aliquots of cDNA were submitted to a subsaturation
number of PCR cycles (40 and 20 cycles for ANP and GAPDH, respectively)
as determined by the sequential cycling experiments.
Amplification of cDNA for ANP and GAPDH was carried out
independently. PCR products of ANP and GAPDH were 320 bp and 297 bp, respectively. Samples were then electrophoresed in 1.5% agarose gels
containing ethidium bromide and quantified via computer densitometry.
Myocyte Isolation and Measurements.
Ten hearts per group
were collected for myocyte isolation, and measurements were made using
methods from this laboratory described previously (Hoque et al., 2000).
In brief, hearts were removed and perfused immediately with
Ca2+-free HEPES-buffered solution for 3 min
followed by 10 min of perfusion with one containing 1.66 mg/ml
collagenase type II (Worthington Biochemical Corp, Lakewood, NJ) and
0.1 mg/ml protease type XIV (Sigma-Aldrich Canada Ltd). The collagenase
was then washed out with a solution containing 0.2 mM
CaCl2. The heart was removed and then separated
into the RV free wall and LV. The left and right ventricular tissues
were separately chopped with scissors and incubated in buffer with 0.2 mM CaCl2 at 37°C for 15 min, filtered, and
centrifuged for 45 s. A cell aliquot was transferred to a stage of
an inverted microscope (Leica Microsystem, Wetzler GmbH, Germany)
equipped with a digital camera (model PDMC-2, Polaroid Canada,
Etobicoke, ON, Canada). The 10 images per sample were photographed
using DirectPhoto software (v2.0, Polaroid Canada) at a 200×
magnification and stored in a computer (Dell XPS T500). The cell area
of 10 cells per image was measured using Mocha software (version 1.2, SPSS Science, Chicago, IL). An average was calculated for each of the
50 cells analyzed for each sample.
Measurement of Pulmonary Artery Wall Thickness.
The lungs
were collected immediately after the animals were sacrificed. Blood was
washed out by 0.9% saline, and lungs were gently dried with paper
towels. The samples were then submerged in 10% buffered formalin for
at least 48 h before which the right lower lobe was embedded in
paraffin. Elastin stains were performed on sections 5 µm thick. The
degree of pulmonary vascular remodeling was assessed by measuring the
percentage of wall thickness of terminal small arteries (vessels
indexed to respiratory bronchioles). At least 10 horizontally cut
terminal small arteries were photographed using a Leica inverted
microscope equipped with a Polaroid digital camera as described for
myocytes. The images were stored on the hard drive of a Dell computer
system and analyzed using Mocha software (SPSS Science). Orthogonal
intercept measurement modified from method described previously
(Gundersen et al., 1978; Jensen et al., 1979) was used to generate 10 random measurements of medial wall thickness (the distance between the
internal and external lamina) and 10 random measurements of diameter of
the vessels (distance between the external lamina). Harmonic means were
taken for each vessel, and an average was calculated for each of the 10 vessels analyzed for each sample. Percentage of wall thickness was
expressed as the medial wall thickness divided by the diameter of the
vessel × 100.
Statistical Analysis. Analyses were performed using SigmaStat (SPSS Science). The variables between groups were compared by analysis of variance and subsequent multiple comparisons by the Student-Newman-Keuls test. A value of P < 0.05 was considered statistically significant. All values were expressed as mean ± S.E.M.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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As shown in Table 1, animals treated
with MCT exhibited significantly depressed body weight gains over the
3-week follow-up period. However, although body weight gain was still
significantly lower from control, MCT-treated animals treated with
cariporide demonstrated a 2-fold higher increase in body weight over
the 3-week period compared with untreated animals. Neither heart rates nor mean arterial pressures were affected by any treatment.
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RV Tissue Weights and Cell Size.
These results are summarized
in Fig. 1 and demonstrate significant
increases in RV weight and cell area following MCT treatment without
cariporide with no direct effect of cariporide itself in the absence of
MCT. The relative increases in RV weight and cell size were 65 and
52%, respectively, in rats that did not receive cariporide. The change
in cell area in the presence of cariporide was 21%. RV weight was
actually 12% less in hearts from the MCT-cariporide group, although
neither tissue weight nor cell size were significantly changed. There
were no significant changes in either LV weight or LV myocyte size with
any treatment (data not shown).
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RV Hemodynamic Responses.
To further determine the degree of
RV responses to MCT treatment, we measured intraventricular pressures
as summarized in Fig. 2. RV systolic
pressures were significantly elevated by 70% in MCT-treated rats,
although the degree of elevation was significantly attenuated by
cariporide to 33%. Moreover, as shown in Fig. 2, the relative
elevation in RV end diastolic pressures was similarly markedly
attenuated in cariporide-treated animals. Thus, RV end diastolic
pressure increased by 94% (P < 0.001) in MCT-treated animals without cariporide, whereas with cariporide this elevation was
attenuated to 42% (P < 0.05).
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RV ANP and NHE-1 mRNA Expression.
MCT treatment was associated
with increased RV expression of ANP as well as NHE-1 mRNA. Compared
with tissues of sham (neither MCT or cariporide) animals, NHE-1 mRNA
increased 2-fold with MCT treatment (P < 0.05),
although this was completely abrogated by cariporide (Fig.
3). MCT treatment also resulted in a
4-fold elevation in ANP expression; however, this was prevented by
cariporide (Fig. 3). There were no changes in either NHE-1 or ANP LV
expression with any treatment (not shown).
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Heart Failure.
A major hallmark associated with
MCT-induced RV responses is the development of heart failure. To
document and characterize more precisely the degree of heart failure,
both cardiac output and central venous pressures were determined. The
degree of heart failure was profound, as evidenced by a 28% reduction
in cardiac output in MCT-treated rats without cariporide
(P < 0.001), whereas in the presence of the NHE-1
inhibitor the reduction in cardiac output was significantly attenuated
to 8%, although the degree of reduction was significant (Fig.
4).
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Myocardial Morphology.
In terms of myocardial tissues, no
abnormalities were observed in hearts of animals not treated with MCT.
However, 8 of 10 RV samples from MCT-treated animals showed the
presence of focal myocyte necrosis and mononuclear leukocyte
infiltration as well as early fibrosis. We observed no histological
abnormalities in MCT-treated animals given cariporide. Representative
micrographs are shown in Fig. 6. No LV
changes were observed in any group (not shown).
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Pulmonary Vascular Morphology.
Because the primary cause of
MCT-induced myocardial complications reflects initial effects of this
agent on pulmonary vessel remodeling, we determined whether the
salutary effects of cariporide reflected an effect on the initial
pulmonary response of MCT or whether cariporide's effect was
attributable to cardiospecific influences. As noted under
Discussion, this is of further interest as there is evidence
that NHE may be involved in vascular remodeling responses to various
factors. As summarized in Fig. 7, MCT
produced a significant (39%) increase in wall thickening. However, in
contrast to the inhibitory effects of cariporide on cardiac responses, the degree of medial wall thickness was virtually identical (41%) to
that seen without cariporide.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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It is well established that NHE-1 inhibition protects the acutely
ischemic and reperfused myocardium (Pierce and Czubryt, 1995; Karmazyn
et al., 1999); indeed, this approach appears to be the most effective
cardioprotective strategy today (Gumina et al., 1999; Gumina and Gross,
1999). Emerging evidence suggests that this antiporter may also be
important in tissue hypertrophy in response to a variety of stimuli.
Accordingly, the primary goal of this study was to evaluate the
potential contribution of the NHE to compensatory myocardial
hypertrophic and subsequent responses that occur after pulmonary
hypertension. The hypothesis that NHE may be involved in these
processes stems from both in vitro and in vivo studies which have shown
that NHE inhibition results in diminished hypertrophy in cardiac
myocytes in response to hypertrophic factors or following left coronary
artery ligation (Hori et al., 1990; Yamazaki et al., 1998; Cingolani,
1999; Karmazyn et al., 1999; Yoshida and Karmazyn, 2000; Kusumoto et
al., 2001). NHE may also be involved in vascular hypertrophy. For
example, neointimal thickening following arterial injury in rats has
been shown to be attenuated by NHE inhibitors (Kranzhöfer et al., 1993). Moreover, cariporide has recently been shown to reduce mesenteric artery remodeling and hypertrophy in the diabetic rat (Jandeleit-Dahm et al., 2000). Thus, taken together, NHE inhibition could represent an effective approach toward minimizing hypertrophic pathological processes. Our results are the first to demonstrate that
NHE-1 inhibition reduces myocardial responses to pulmonary artery
injury. They further suggest that these effects occur independent of
any influence on the primary pulmonary artery responses.
We used the MCT model of pulmonary hypertension because this approach
has been well characterized and resembles clinical pulmonary vascular
disease (Rosenberg and Rabinovitch, 1998; Schultze and Roth, 1998;
Nakazawa et al., 1999). MCT produces its effects primarily by injuring
pulmonary vessel endothelium, resulting in reduced vasodilating
responses and increased vascular resistance (Schultze and Roth, 1998).
Blocking the pulmonary hypertension represents a key therapeutic
approach for the deleterious effects of MCT in rats and clearly is of
importance in developing effective strategies for this life-threatening condition.
As noted above, we hypothesized that provision of a NHE-1-specific
inhibitor should diminish both the vascular and myocardial responses to
MCT since NHE-1 is the primary NHE isoform in both myocardium as well
as vascular smooth muscle cells. However, our findings demonstrate that
pulmonary artery remodeling as determined by vessel thickness after MCT
treatment was unaffected by cariporide. In view of the studies referred
to above suggesting that NHE-1 may also be important in vascular
remodeling (Kranzhöfer et al., 1993; Jandeleit-Dahm et al.,
2000), we envisaged that cariporide administration would also probably
have reduced pulmonary artery remodeling. The failure to observe this
may reflect a number of factors, including differences in stimuli to
induce vascular hypertrophy or tissue-specific differences in
NHE-1-dependent vascular responses. With respect to the latter, note
that inhibition of smooth muscle cell proliferation by NHE-1 inhibitors
has not been demonstrated in pulmonary arteries, thus raising the
possibility that these vessels undergo remodeling through a
NHE-1-independent pathway.
Our results therefore demonstrate that the effect of cariporide is
restricted to direct inhibition of the compensatory myocardial responses to MCT-induced resultant pulmonary vascular remodeling. The
beneficial effects were clearly observed with respect to a variety of
indices, including reduced RV hypertrophy and complete abrogation of
morphological abnormalities as well as improved function including
attenuation of heart failure. The attenuation of RV hypertrophy was
documented by using multifaceted approaches, including measurement of
cell size, tissue gravimetric analysis, and determination of expression
of ANP, which is known to be increased in the hypertrophied myocardium
(Durocher et al., 1998). Moreover, NHE-1 RV mRNA expression was also
elevated in response to MCT, albeit to a lesser degree than ANP,
although this was prevented by cariporide. Although the results
indicate an up-regulation of both genes, note that mRNA expression was
done using semiquantitative approaches, which limits detailed
interpretation of the data. Weight gain, which has been shown to be
greatly reduced after MCT administration (Kodama and Adachi,
1999), was twice that in the cariporide group treated with MCT when
compared with MCT alone, further demonstrating that animals on
cariporide treatment fared better than their untreated counterparts,
further suggesting a marked reduction in morbidity in these animals.
The precise mechanism for the salutary effect of cariporide on
compensatory myocardial response needs to be determined especially in
view of the latter's complex nature. For example, RV hypertrophy following MCT treatment is associated with increased oxidative stress
(Pichardo et al., 1999), elevations in both 
1-
and 
-adrenergic receptor densities (Chen et al., 1999), reduced
natriuretic peptide receptors (Kim et al., 1999), up-regulation of the
endothelin system (Ueno et al., 1999), electrophysiological changes
(Lee et al., 1997), as well as the dispersion and disorganization of the principal gap junction protein connexin 43 (Uzzaman et al., 2000).
Therefore, the RV hypertrophy following MCT treatment is undoubtedly a
complex phenomenon. As referred to above, the role of NHE-1 probably
lies in its ability to contribute to the hypertrophic process, but how
this occurs is unknown. Recent evidence suggests that sodium ions may
represent a key factor. In this regard, note that NHE-1 is a
significant source of sodium entry into the cardiac cell and that
sodium ions are known to be important mediators of cell hypertrophy (Gu
et al., 1999; Hayasaki-Kajiwara et al., 1999). One hypothesis suggests
that the role of sodium reflects its ability to activate distinct PKC
isoforms, especially PKC
and PKC
, resulting in increased protein
synthesis and hypertrophy (Hayasaki-Kajiwara et al., 1999). Other
hypotheses have also been proposed, including the NHE-1-dependent
activation of both Raf-1 and MAP kinase resulting in cardiomyocyte
hypertrophy (Yamazaki et al., 1998). Further studies are necessary to
identify the precise mechanisms(s) underlying what appears to be a
critical role for NHE-1 in RV hypertrophy as well as the antiporter's
role in myocardial remodeling in general.
In conclusion, our study demonstrates that a NHE-1-selective inhibitor, cariporide, attenuates the RV hypertrophy and heart failure after MCT administration in rats in the absence of effect of pulmonary vessel remodeling. Taken together, the results suggest an antihypertrophic effect of the drug on the myocardium as well as a contributory role of NHE-1 in the myocardial adaptive response to pulmonary vascular injury in this model. Our results suggest that in principle, NHE-1 inhibition represents a desirable approach to reduce the RV involvement in pulmonary hypertension, particularly in conjunction with other therapies aimed at targeting the primary pulmonary artery dysfunction.
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Acknowledgments |
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We thank Aventis Pharma (Frankfurt, Germany) for providing us with experimental diets and for performing analysis of serum cariporide levels.
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Footnotes |
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Accepted for publication May 1, 2001.
Received for publication February 14, 2001.
This study was funded by a grant from the Canadian Institutes of Health Research. Dr. Karmazyn is a Career Investigator of the Heart and Stroke Foundation of Ontario.
Address correspondence to: Dr. Morris Karmazyn, Department of Pharmacology and Toxicology, University of Western Ontario, Medical Sciences Building, London, Ontario N6A 5C1 Canada. E-mail: mkarm{at}uwo.ca
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Abbreviations |
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RV, right ventricle; ANP, atrial natriuretic peptide; MCT, monocrotaline; NHE-1, Na+-H+ exchange isoform 1; LV, left ventricle; RT-PCR, reverse transcriptase-polymerase chain reaction; dNTP, deoxynucleotide triphosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PKC, protein kinase C; DEPC, diethyl pyrocarbonate.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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and in cultured neonatal rat cardiac myocytes.
J Mol Cell Cardiol
31:
1559-1572[Medline].1-adrenergic-mediated responses in rat ventricular myocytes by adenosine A1 receptor activation: role of the KATP channel.
J Pharmacol Exp Ther
294:
770-777
the monocrotaline model and involvement of the hemostatic system.
J Toxicol Environ Health B Crit Rev
1:
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F. Akhavein, E. J. St.-Michel, E. Seifert, and C. V. Rohlicek Decreased left ventricular function, myocarditis, and coronary arteriolar medial thickening following monocrotaline administration in adult rats J Appl Physiol, July 1, 2007; 103(1): 287 - 295. [Abstract] [Full Text] [PDF]
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A. Baartscheer, C. A. Schumacher, M. M.G.J. van Borren, C. N.W. Belterman, R. Coronel, T. Opthof, and J. W.T. Fiolet Chronic inhibition of Na+/H+-exchanger attenuates cardiac hypertrophy and prevents cellular remodeling in heart failure Cardiovasc Res, January 1, 2005; 65(1): 83 - 92. [Abstract] [Full Text] [PDF]
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 Y. V. Mukhin, M. N. Garnovskaya, M. E. Ullian, and J. R. Raymond ERK Is Regulated by Sodium-Proton Exchanger in Rat Aortic Vascular Smooth Muscle Cells J. Biol. Chem., January 16, 2004; 279(3): 1845 - 1852. [Abstract] [Full Text] [PDF]
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L. Chen, C. X. Chen, X. T. Gan, N. Beier, W. Scholz, and M. Karmazyn Inhibition and reversal of myocardial infarction-induced hypertrophy and heart failure by NHE-1 inhibition Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H381 - H387. [Abstract] [Full Text] [PDF]
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M. Avkiran and R. S Haworth Regulatory effects of G protein-coupled receptors on cardiac sarcolemmal Na+/H+ exchanger activity: signalling and significance Cardiovasc Res, March 15, 2003; 57(4): 942 - 952. [Abstract] [Full Text] [PDF]
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X. Li, A. J. Misik, C. V. Rieder, R. J. Solaro, A. Lowen, and L. Fliegel Thyroid Hormone Receptor alpha 1 Regulates Expression of the Na+/H+ Exchanger (NHE1) J. Biol. Chem., August 2, 2002; 277(32): 28656 - 28662. [Abstract] [Full Text] [PDF]
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C. V. Rieder and L. Fliegel Developmental regulation of Na+/H+ exchanger expression in fetal and neonatal mice Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H273 - H283. [Abstract] [Full Text] [PDF]
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P < 0.05 from respective MCT
untreated group. Numbers in parentheses indicate number of samples
studied; however, for cell measurements 50 cells were analyzed and
averaged to obtain one value. Photographs at bottom show examples of RV
myocytes from sham untreated animals (A), MCT untreated animals (B),
and MCT cariporide animals (C). Scale bar, 100 µm. bw, body weight.
) or vehicle (control,
) administration. Values are means ± S.E.M.
**P < 0.01, ***P < 0.001 from
respective control group; 
