Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
Ouabain is a steroid hormone which is released from the
hypothalamus and the adrenal gland. Ouabain and other
cardiac glycosides are known as specific inhibitors of
sodium pumps ever since the very first observations of the
inhibition of sodium pumps of red blood cells. As a sodium
pump is an intrinsic plasma membrane enzyme that
hydrolyses ATP to maintain the transmembrane gradients of
Na+ and K+ found in most mammalian cells, as an inhibitor of sodium
pumps, ouabain is implicated in sodium homeostasis and
exerts direct actions on the vasculature, the
heart[1], and tubular sodium
reabsorption[2], thus playing an important role
in the pathogenesis of hypertension and some other
cardiovascular disorders.
There are many data suggesting that ouabain is
associated with both high blood pressure and altered cardiac
morphology in hypertensive
patients[3,4]. For patients with more
advanced hypertension, circulating levels of ouabain were
directly related to both blood pressure and total peripheral
resistance, and inversely related to cardiac
index[4]. The presence of left ventricular hypertrophy with high plasma
ouabain levels has been found to be associated with alterations
in cardiac function[5]. The young offspring of hypertensive
patients had higher plasma levels of ouabain than the
offspring of normotensive parents, which were correlated with
diastolic dysfunction[6]. Moreover, circulating ouabain is a
novel, independent, and incremental marker that predicts the
progression of heart failure according to Pitzalis's
report[7].
Although ouabain might have a primary role in causing
cardiac dysfunction and failure, its precise role and
mechanism remains unclear. Our research group proposed that
ouabain at pathological concentrations might affect the
structure and function of the vascular endothelium and trigger
vascular remodeling in hypertension[8]. Due to the
demonstrated effects of ouabain on vascular areas, an effect of
ouabain on cardiac remodeling and function could be
predicted, so it prompted us to investigate ouabain's
contributions to the heart, on the assumption that ouabain might
have a primary role in the progression of both vascular
remodeling and cardiac remodeling in hypertension. This study
investigated ouabain's effects on the rat heart in order to
assess whether the steroid hormone affects the structure
and function of the heart and if it is involved in cardiac
remodeling.
Materials and methods
Reagents and drugs Ouabain was purchased from Sigma
(St Louis, MO, USA). Kits for RNA extraction and reverse
transcription were purchased from Invitrogen (San Diego,
CA, USA). The primers and probes were synthesized by
Sangon Biological Engineering Technology and Service Co
(Shanghai, China). All other reagents were of analytical grade.
Established animal model The rats used to establish the
animal model were Grade II (Certificate No 08-005). The
project was approved by the Animal Experimentation Ethics
Committee of Xi'an Jiaotong University (Xi'an, China). For
the experiments, 6_10-week-old male Sprague-Dawley rats
(150_200 g) were used after being kept in a
temperature-controlled room for a week. The rats were randomly
assigned to the experimental and control groups. The rats in
the former group (n=48) received a maintenance dose of
ouabain intra-peritoneally. On d 1, a loading dose of 34 mg/kg ip
was administered, followed by a dose of 27.8
mg·kg-1·d-1 ip for an additional 6 weeks every day. The control group
(n=40) received vehicle (0.9% saline) only. Ouabain was
dissolved in sterile saline at a concentration of 20 mg/mL
and stored for up to 1 week at 4 oC in the dark. Systolic blood
pressure (SBP) was recorded with a tail cuff once a week
using a heart rate and blood pressure recorder for rats
(RBP-1B; China-Japan Clinical Medicine Institute, Beijing, China).
The average of 3 recordings was taken as the individual
SBP. Measurements obtained using this method correlated
well with those obtained using the direct cannulation
method[9]. The 8 rats whose blood pressure was unchanged by
ouabain treatment ("ouabain-resistant"
rats[8]) were excluded. The 2 groups of animals were kept in the same conditions
during the experiment. At the end of 4 and 6 weeks,
echo-cardiography was performed, hemodynamic parameters were
measured by invasive cardiac catheterization, and then the
animals were sacrificed and the apical half of the ventricular
tissues was collected for the experiments.
Echocardiographic examination Transthoracic
echo-cardiography was performed on the anaesthetized rats
using a Sonos 5500 echocardiographic system (Philips
Medical Systems, Andover, MA, USA). The system was
equipped with a 7-12 MHz transducer that was placed on the shaved
left hemithorax. The parasternal long-axis views of the left
ventricle at or just below the tip of the mitral valve leaflets
were used to obtain targeted M-mode recordings. The left
ventricular end-diastolic diameter (LVEDd) and end-systolic
diameter (LVEDs), septal thickness (IVST) and posterior wall
thickness (PWT) were measured. The pulsed Doppler
recordings were made from the standard apical 4 chamber view.
Some indexes of left ventricular filling by Doppler flow
velocity were measured: the isovolumic relaxation time (IVRT),
the peak velocity of early left ventricular filling (E), the peak
velocity of late ventricular filling (A), and the ratio between
the early and late peak flow velocity (E/A). All data given are
the means for 5 consecutive cardiac cycles and all
measurements were manually obtained by the same observer. Midwall
fractional shortening (FS) was calculated as describ-ed by
Shimizu et al[10]. The ejection fraction (EF) was measured
using a modified version of Simpson's monoplane
analysis[11].
Hemodynamic measurements Hemodynamics data were
obtained by catheterization of the right common carotid
artery as described previously[12]. The carotid artery was
isolated and cannulated with a 3-F high-fidelity microtip
catheter connected through a data acquisition unit (PowerLab
4.12, AD Instruments Inc, Castle Hill, NSW, Australia) to a
computer running MacLab software (AD Instruments Inc,
Australia). The catheter was advanced into the left ventricle.
After an equilibration period of 20 min, the left ventricular
systolic pressures (LVSP) and end-diastolic pressures
(LVEDP), and the left ventricular
dp/dtmax were monitored.
All hemodynamic parameters were recorded continuously
for 15 min. Then the rats were sacrificed and the hearts were
isolated rapidly. The left ventricles were cut into 2 pieces;
one piece was fixed with 4% paraformaldehyde for
histo-chemistry, and the other was stored in liquid nitrogen for
real-time quantitative RT-PCR.
Myocardial ultrastructure examination The animals were
anesthetized with 20% urethane ip, and the apical part of the
ventricle was removed. The samples were chipped into
tissue blocks of 1 mm3 on ice and immediately put into a 2.5%
glutaraldehyde fixation solution at 4 oC for 2 h. Electron
microscope slices were produced according to routine
methods for the preparation of transmission electron microscope
specimens. Ultrathin sections were stained with uranyl
acetate and lead citrate for examination on a transmission
electron microscope (Hitachi H600, Tokyo, Japan).
Morphological examination The samples of the left
ventricle were fixed in 4% paraformaldehyde for 24 h, dehydrated
in ethanol, cleared in dimethylbenzene, and embedded in
paraffin. The sections were prepared and stained with
Picrosirius red[13]. The slides were observed with a Nikon
Microphot FXA light microscope (Tokyo, Japan) equipped
with a polarized set. The collagen fraction of the left
ventricle (CFLV) was assessed using Picrosirius red stain and
calculated as described previously[14]. The CFLV was
expressed as the mean percentage of connective tissue areas
divided by the total tissue area in the same field. The
operator was blinded to the experimental group during the analysis.
RT-PCR primers and probes design Primers and probes
for quantitative RT-PCR were designed and synthesized by
Sangon Biological Engineering Technology and Service Co
(Shanghai, China). The melting temperature for the primers
was set at 50-60 oC, but the probe's melting temperature was
at least 10 oC higher. The minimum GC content for the
primers and probes was 20%-80%, and runs of identical
nucleotides were avoided. All the probes were labeled at the 5'
end with the reporter dye molecule, FAM
(6-carboxy-fluorescein), and at the 3' end with the quencher dye, TAMRA
(6-carboxytetramethylrodamine; Table 1).
Quantitative RT-PCR The total RNA in the left
ventricles was extracted using the Trizol Max kit (Invitrogen,
USA). The expression of MHC-α and MHC-β was examined
by fluorescent real-time quantitative RT-PCR. The
quantitative RT-PCR was performed using SuperScript one-step
RT-PCR kit with the Platinum Taq system (Invitrogen, USA),
following the manufacturer's instructions (with
modifica-tions). The cDNA amplification product was predicted to be
a 164 bp fragment for MHC-α, and a 148 bp fragment for
MHC-β. A total reaction volume consisting of 1 µL forward
primer, 1 µL reverse primer, 1 µL fluorescent probe, and 1 µL
RT/Platinum Taq Mix, and 4 µL total RNA was made up to 50
µL with RNase-free water. One-step RT-PCR amplification
was performed in the ABI Prism 7700 Sequence Detection
System (Perkin-Elmer, Foster City, CA, USA). Thermal
cycling conditions were as follows: 94
oC for 5 min, followed by 50 cycles of 94
oC for 30 s, 56 oC for 30 s, and 72
oC for 30 s, then 72 oC for 5 min, and finally the reaction was held at 4
oC. The housekeeping gene GAPDH was used to normalize
samples to remove the impact of any variations in RNA
loading. The
2—ΔΔCT method was used to calculate relative
changes in gene expression determined from the real-time
quantitative RT-PCR experiment[15].
Statistical analysis Data are expressed as mean±SD.
Statistical differences were determined by ANOVA with the
SPSS software package (version 10.0). Student's
t-test was employed to compare the data between the 2 groups. A
probability value of P<0.05 was considered statistically
significant.
Results
Effects of ouabain on SBP In the 4 weeks, there was no
significant difference in the mean SBP between the ouabain
group and the control group (P>0.05). After 4 weeks, the
mean SBP in the ouabain group began to increase and was
significantly higher than that in control group after 6 weeks
(P<0.01; Figure 1).
Echocardiographic findings Four weeks after treatment
with ouabain, the rats in the ouabain group showed clear
signs of left ventricle dysfunction, whereas the rats in the
control group displayed normal echocardiographic parameters. Systolic performance worsened in the ouabain
group, which is indicated by the reduced EF and FS.
Diastolic performance was affected also, as shown by the
decreased E/A and increased IVRT. LVEDs, LVEDd, PWT and
IVST were significantly greater in the rats of the ouabain
group, indicating that left ventricle enlargement and left
ventricle wall thickening were induced by ouabain treatment
(Table 2; Figure 2). Six weeks after treatment with ouabain,
echocardiographic parameters of the rats were more severe
than that of the rats treated by ouabain for 4 weeks.
Hemodynamic data After treatment with ouabain for 4
weeks, there were no significant differences in the carotid
blood pressure (CBP) of the 2 groups. However, compared
with the normal control animals, LVSP and
±dp/dt significantly decreased and LVEDP increased in the rats of the
ouabain group. After treatment with ouabain for 6 weeks,
the CBP in the ouabain group was significantly higher than
that in the control group, and all hemodynamic results
further deteriorated (Table 3).
Changes in myocardial ultrastructure Cardiac muscle
fibers in the control group were abundant, with regular
arrays of myofibrils closely arranged within the sarcomere.
The mitochondria were intact and had no swelling or
disruption. There were little collagen fibers between the
myocardial cells in the control group. Cardiac muscle fibers,
mitochondria, and collagen fibers in the ouabain group were
significantly different from those in the control group. The
differences included reduction of the sarcoplasm content,
disorganization of myofilaments and Z line, mitochondrial
swelling, disruption and vacuolation, and hyperplastic
collagen fibers. The differences were obvious in the rats treated
with ouabain for 4 weeks (Figure 3).
Morphometric histology After treatment with ouabain
for 4 weeks, Picrosirius red observation under polarized light
showed that CFLV significantly increased in the rats of the
ouabain group, which indicated that the collagen
metabolism in left ventricle was affected by ouabain treatment [at 4
weeks 5.97±0.39 (ouabain group)
vs 4.92±0.50 (control group); at 6 weeks 6.38±0.53
vs 5.05±0.62, P<0.05].
Expression of MHC-α and MHC-β mRNA in the left
ventricles After treatment with ouabain for 4 weeks, real-time
quantitative RT-PCR showed a 0.74× fold decrease (0.65_0.83, CV 10.81%) of MHC-α mRNA expression in the
ouabain-treated rats compared with the vehicle rats. In contrast,
MHC-β mRNA expression increased 2.26× fold (1.93_2.58,
CV 14.60%). Six weeks after treatment with ouabain,
MHC-α mRNA expression decreased 0.55×fold (0.43_0.67, CV 21.82%),
and MHC-β mRNA increased 3.28×fold (2.83_3.73, CV
13.41%) compared with the control group (Figure 5).
Discussion
It has been demonstrated that ouabain administered ip is
readily absorbed, and that plasma ouabain levels are not
significantly different between intraperitoneal and
intravenous groups at 10 min after
administration[16]. The doses of ouabain infused in our study were determined based on
previously published pharmacokinetic data for ouabain in rats.
The doses administered were estimated to increase average
plasma levels 0.5_1.0 nmol/L above the physical
level[17] (the physical concentration of plasma ouabain is approximately
0.3_0.9 nmol/L[18]), so the plasma ouabain concentration in
the animals is similar to the levels present in many
pathological states such as hypertension and congestive heart failure
(the pathological concentration of plasma ouabain is
approximately 0.9_1.8 nmol/L[18]). Moreover, based on the animals'
weight gain, behavioral changes, and cardiac rhythms, the
effects of ouabain did not appear to be associated with
substantial toxicity.
In this study, we investigated ouabain's effects on
cardiac remodeling in rats. The primary findings are as follows:
left ventricular hypertrophy, myocardial ultrastructure
deterioration, and extracellular matrix remodeling were
induced by ouabain treatment; meanwhile, cardiac systolic
and diastolic performance both worsened. Moreover, the
cardiac MHC-β mRNA, a marker of hypertrophy or failure,
was upregulated by ouabain treatment, whereas
MHC-α mRNA was downregulated. The effects of ouabain were
found before the increase of blood pressure, indicating that
ouabain might damage the structure and function of the rat
heart and be involved in cardiac remodeling independent of
blood pressure.
There are other studies providing evidence that ouabain
may cause pathological cardiac hypertrophy independent
of blood pressure. In a study in rats, a chronic infusion of
very low doses of ouabain to double the plasma
concentration of ouabain triggered a signal transduction pathway that
produced cardiac hypertrophy[5]. Another study showed
that partial inhibition of Na+/K+
ATPase by ouabain causes hypertrophic growth and transcriptional regulation of
several growth-related marker genes in cultured neonatal rat
cardiac myocytes[19].
However, how does ouabain exert such effects without a
change in blood pressure? Recent studies have provided
novel insights into ouabain's role. When ouabain binds to
Na+/K+ ATPase, it converts the enzyme to a signal
transducer and initiates multiple gene regulatory
pathways[19]; the cellular effects of ouabain on the heart may include
actions independent of Na+/K+
ATPase inhibition[20]. We suppose that the mechanisms underlying these actions of
ouabain are as follows: first, ouabain interaction with
Na+/K+ ATPase initiates multiple growth-related gene regulatory
pathways, triggers growth and the proliferation of cardiac
myocytes and fibroblasts. It is
reported[19] that ouabain's effects includes the activation of the Src kinase and tyrosine
phosphorylation of the epidermal growth factor receptors
and other proteins, followed by the activation of Ras, the
Ras/Raf/MEK//MAPK cascade, and increased production
of reactive oxygen species. Thus, cardiac hypertrophy and
extracellular matrix remodeling are induced by ouabain
treatment. Second, ouabain is a steroid hormone in nature; it
has a complicated interaction with other neurohormonal
systems, such as the renin-angiotensin and the endothelin
system[21]. Saunders and Scheiner-Bobis suggest that
ouabain stimulates endothelin release and expression in human
endothelial cells[22]. Our research group previously found
that plasma angiotensin (Ang) II and endothelin were
increased by ouabain treatment in
rats[23,24]. It is well known that Ang II and endothelin have toxic effects on cardiac
myocytes and play an important role in the development of
pathological cardiac remodeling. Ouabain's effects on the
renin-angiotensin and the endothelin system might be
involved in the damage of cardiac structure and function.
Echocardiography is one of the most widely used,
noninvasive techniques to provide quantitative
measurements of ventricular structure and function in humans and
experimental animals. In rats, the heart is generally 1 g in
weight with a 2 mm left ventricle wall thickness and a
rapid heart rate of 300_400 bpm. Conventional transthoracic
echocardiography transducers often do not provide enough
resolution. However, high-frequency transducers (7.5_15
MHz) are now available to monitor morphometric and
functional changes in small animals in
vivo[25]. Hemodynamics monitoring is one of the reliable methods to evaluate left
ventricle performance by invasive catheterization. We chose
the 2 kinds of techniques to evaluate ouabain's effects on
the function of the rat heart.
Our results suggested that systolic and diastolic
performance both worsened with ouabain treatment. Hypertrophic
left ventricle and hyperplastic collagen fibers might be the
reasons of diastolic dysfunction. The reasons for systolic
dysfunction are multiple. At first our study showed that the
mitochondria were damaged by ouabain treatment, and
deteriorated energy metabolism could be predicted. Ouabain
might affect systolic performance through disturbing the
energy metabolism of cardiac myocytes. Second, the
hypertrophy-associated switch of adult and fetal isoforms of
myosin heavy chain expression might contribute to the systolic
dysfunction in the ouabain-treated rats. In normal young
adult rats, 80%_90% of the myosin heavy chain expressed is
the α isoform[26]. The MHC-α is associated with higher
ATPase activity, and so hearts rich in MHC-α have a high
intrinsic contractility. In contrast, MHC-β is associated with
low ATPase activity and a low intrinsic contractility. As
showed in this study, decreased MHC-α mRNA expression
and increased MHC-β mRNA expression were induced by
ouabain treatment, so the animal's cardiac systolic
performance was affected. Finally, as an inhibitor of
Na+/K+ ATPase, a short-term administration of ouabain can exert a
positive inotropic effect on cardio-myocytes by inhibiting
the plasma membrane Na+/K+ ATPase, increasing the
intracellular Ca-concentration and decreasing the Ca-extrusion by
the sodium/calcium exchanger (NCX). However, there are
data suggesting that long-term administration of ouabain
has no effect on myocardial contractility in rats. According
to Muller-Ehmsen et al[27], chronic ouabain treatment
increases the protein expression of the NCX, and the positive
inotropic effect can no longer be observed after chronic
treatment for 2 d. Further more, NCX overexpression impairs
ouabain-dependent cell shortening in adult rat
cardiomyo-cytes[28]. In this study, chronic ouabain treatment for 4 or 6
weeks might induce NCX overexpression, and myocardial
contractility might be impaired subsequently; this may be
one of the ways through which ouabain affects systolic
performance.
In conclusion, our study provides evidence that
ouabain might damage the structure and function of the rat heart
independent of blood pressure. Ouabain does not only
trigger mechanisms initiating primary hypertension, but also
plays an important role in the development of cardiac
remodeling. Clinical research during the past several
decades has shown the importance of cardiac remodeling as a
basic mechanism in the progression of heart failure. In the
future, these novel insights into the role of ouabain in
cardiac remodeling might allow the development of novel
therapeutic strategies to treat cardiac remodeling and failure.
Acknowledgement
We would like to thank all the other members of our
ouabain research group for their suggestions and technical
assistance.
References
1 Schoner W. Endogenous cardiac glycosides, a new class of
steroid hormones. Eur J Biochem 2002; 269: 2440_8.
2 Manunta P, Messaggio E, Ballabeni C, Sciarrone MT, Lanzanic
C, Ferrandi M, et al. Plasma ouabain-like factor during acute and
chronic changes in sodium balance in essential hypertension.
Hypertension 2001; 38: 198_203.
3 Manunta P, Stella P, Rivera R, Ciurlino D, Cusi D, Ferrandi M,
et al. Left ventricular mass, stroke volume, and ouabain-like factor
in essential hypertension. Hypertension 1999; 34: 450_6.
4 Pierdomenico SD, Bucci A, Manunta P, Rivera R, Ferrandi M,
Hamlyn JM, et al. Endogenous ouabain and hemodynamic and
left ventricular geometric patterns in essential hypertension.
Am J Hypertens 2001; 14: 44_50.
5 Ferrandi M, Molinari I, Barassi P, Minotti E, Bianchi G, Ferrari
P. Organ hypertrophic signaling within caveolae membrane
subdomains triggered by ouabain and antagonized by PST 2238. J
Biol Chem 2004; 279: 33 306_14.
6 Manunta P, Iacoviello M, Forleo C, Messaggio E, Hamlyn JM,
Lucarelli K, et al. High circulating levels of endogenous ouabain
in the offspring of hypertensive and normotensive individuals. J
Hypertens 2005; 23: 1677_81.
7 Pitzalis MV, Hamlyn JM, Messaggio E, Iacoviello M, Forleo C,
Romito R, et al. Independent and incremental prognostic value
of endogenous ouabain in idiopathic dilated cardiomyopathy. Eur
J Heart Fail 2006; 8: 179_86.
8 Ren YP, Huang RW, Lv ZR. Ouabain at pathological
concentrations might induce damage in human vascular endothelial cells.
Acta Pharmacol Sin 2006; 27: 165_72.
9 Tipton CM, Sebastian LA, Overton JM, Woodman CR, Williams
SB. Chronic exercise and its hemodynamic influences on resting
blood of hypertension rats. J Appl Physiol 1991; 71: 2206_11.
10 Shimizu G, Hirota Y, Kita Y, Kawamura K, Saito T, Gaasch WH.
Left ventricular midwall mechanics in systemic arterial
hyper-tension. Myocardial function is depressed in pressure-overload
hypertrophy. Circulation 1991; 83: 1676_84.
11 Schiller NB, Shah PM, Crawford M, DeMaria A, Devereux R,
Feigenbaum H, et al. Recommendations for quantification of
the left ventricle by two-dimensional echocardiography.
American Society of Echocardiography Committee on Standards,
Subcommittee on quantification of two-dimensional
echocardio-grams. J Am Soc Echocardiogr 1989; 2:
358_67.
12 Hou XW, Son J, Wang Y, Ru YX, Lian Q, Majiti W,
et al. Granulocyte Colony-Stimulating Factor Reduces Cardiomyocyte
Apoptosis and Improves Cardiac Function in Adriamycin-Induced
Cardiomyopathy in Rats. Cardiovasc Drugs Ther 2006; 20:
85_91.
13 Zhang H, Sun L, Wang W, Ma X. Quantitative analysis of
fibrosis formation on the microcapsule surface with the use of
picro-sirius red staining, polarized light microscopy, and digital image
analysis. J Biomed Maser Res A 2006; 76: 120_5.
14 Tao X, Liu GL. Protection of organic trauma in
sinoaortic-denervated rats treated with fosinopril. Acta Pharm Sin 2003;
38: 743_7.
15 Livak KJ, Schmittgen TD. Analysis of relative gene expression
data using real-time quantitative PCR and the
2—ΔΔCT method. Methods 2001; 25: 402_8.
16 Wang H, Yuan WQ, Lu ZR. Differential regulation of the sodium
pump α-subunit isoform gene by ouabain and digoxin in tissues of
rats. Hypertens Res 2000; 23: S55_60.
17 Tian G, Dang CX, Lu ZR. The changes and significance of the
Na+, K+ -ATPase α-subunit in ouabain-hypertensive rats.
Hypertens Res 2001; 24: 729_34
18 Hamlyn JM, Manunta P. Ouabain, digitalis-like factors and
hypertension. J Hypertens 1992; 10: S99_111.
19 Kometiani P, Li J, Gnudi L, Kahn BB, Askari A, Xie ZJ. Multiple
signal transduction pathways link
Na+/K+-ATPase to growth-related genes in cardiac myocytes. The roles of Ras and
mitogen-activated protein kinases. J Biol Chem 1998; 273: 15 249_56.
20 Nishio M, Rush SW, Wasserstrom JA. Positive inotropic effects
of ouabain in isolated cat ventricular myocytes in sodium-free
conditions. Am J Physiol Heart Circ Physiol 2002; 283:
H2045_53.
21 Xavier FE, Yogi A, Callera GE, Tostes RC, Alvarez Y, Salaices
M, et al. Contribution of the endothelin and renin-angiotensin
systems to the vascular changes in rats chronically treated with
ouabain. Br J Pharmacol 2004; 143: 794_802.
22 Saunders R, Scheiner-Bobis G. Ouabain stimulates endothelin
release and expression in human endothelial cells without
inhibiting the sodium pump. Eur J Biochem 2004; 271: 1054_62.
23 Guo N, Jiang X, Lv ZR, Ai WT. The expression of angiotensin II
and its subtype 1 and 2 recepors in the myocardium of
ouabain-induced hypertensive rats. J Xi'an Jiaotong Univ (Med Sci) 2005;
26: 526_9. Chinese.
24 Jiang X, Guo N, Lv ZR, Ai WT. Effects of ouabain on endothelin
system in rat heart. J Xi'an Jiaotong Univ (Med Sci) 2005; 26:
519_22. Chinese.
25 Kokubo M, Uemura A, Matsubara T, Murohara
T. Noninvasive evaluation of the time course of change in cardiac function in
spontaneously hypertensive rats by echocardiography. Hypertens
Res 2005; 28: 601_9.
26 Nakanishi K, Nakata Y, Kanazawa F, Imamura SI, Matsuoka R,
Osada H, et al. Changes in myosin heavy chain and its
localization in rat heart in association with hypobaric hypoxia-induced
pulmonary hypertension. J Pathol 2002; 197: 380_7.
27 Muller-Ehmsen J, Nickel J, Zobel C, Hirsch I, Bolck B, Brixius K,
et al. Longer term effects of ouabain on the contractility of rat
isolated cardiomyocytes and on the expression of Ca and Na
regulating proteins. Basic Res Cardiol 2003; 98: 90_6.
28 Bolck B, Munch G, Mackenstein P, Hellmich M, Hirsch I, Reuter
H, et al. Na+/Ca2+ exchanger overexpression impairs frequency-
and ouabain-dependent cell shortening in adult rat cardiomyocytes.
Am J Physiol Heart Circ Physiol 2004; 287: H1435_45.
|
