Download Physiological and pathological cardiac hypertrophy induce different

Am J Physiol Regulatory Integrative Comp Physiol
281: R2029–R2036, 2001.
Physiological and pathological cardiac hypertrophy
induce different molecular phenotypes in the rat
MOTOYUKI IEMITSU,1 TAKASHI MIYAUCHI,1 SEIJI MAEDA,1
SATOSHI SAKAI,1 TSUTOMU KOBAYASHI,1 NOBUHARU FUJII,2
HITOSHI MIYAZAKI,2 MITSUO MATSUDA,3 AND IWAO YAMAGUCHI1
1
Cardiovascular Division, Department of Internal Medicine, Institute of Clinical
Medicine, 2Gene Experiment Center, Institute of Applied Biochemistry, and 3Institute
of Health and Sport Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan
Received 23 April 2001; accepted in final form 27 July 2001
causes cardiac hypertrophy,
which is defined as athletic heart (7, 26). The athletic
heart is a physiological cardiac hypertrophy, which is
an induced beneficial adaptive response of the cardiovascular system, i.e., decreased resting and submaximal heart rates and increased filling time and venous
return (1, 22, 28, 35). Together, these adaptations can
help the myocardium satisfy the increased demands of
exercise while maintaining or enhancing normal function (1, 22, 28, 35). Although it has been considered
that exercise training-induced cardiac hypertrophy is
partly caused by the increase in mechanical load by
repeated bouts of exercise (28), the precise mechanisms
are not known.
Hypertension and cardiac valvular disease induce
pathological cardiac hypertrophy caused by pressure
overload (24, 28). Pathological cardiac hypertrophy is a
compensatory adaptation to an increase in workload of
the heart (24). Pathological cardiac hypertrophy reduces cardiac function in the left ventricle (28). Furthermore, it has been reported that the progression of
pathological cardiac hypertrophy results in heart failure (24, 27). Thus there are differences in cardiac
properties between pathological and physiological cardiac hypertrophy (athletic heart).
Recently, it has been reported that some cardiovascular regulating factors participate in pathological cardiac hypertrophy (27). Recent in vivo studies have
suggested that ANG II is a growth factor for pathological cardiac hypertrophy (16, 40). ANG II is converted
from ANG I by angiotensin-converting enzyme (ACE).
It has also been reported that ANG II plays an important role in the pathogenesis of heart failure (25).
Furthermore, increased expression of ACE has been
reported in the failing heart (25). Endothelin-1 (ET-1)
also induces cardiac hypertrophy (11, 36). We previously reported that the tissue level of ET-1 is markedly
increased in the failing heart of rats with congestive
heart failure due to myocardial infarction (30, 31).
Activation of the myocardial ␤1-adrenergic pathway
also induces cardiac hypertrophy (27, 42). Furthermore, pressure overload hypertrophy and failing heart
cause an increase in mRNA expression of brain natri-
Address for reprint requests and other correspondence: T. Miyauchi, Cardiovascular Div., Dept. of Internal Medicine, Institute of
Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 3058575, Japan (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
cardiovascular regulating factor; athletic heart; spontaneously hypertensive rat; swim training; hypertension
CHRONIC EXERCISE TRAINING
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Iemitsu, Motoyuki, Takashi Miyauchi, Seiji Maeda,
Satoshi Sakai, Tsutomu Kobayashi, Nobuharu Fujii,
Hitoshi Miyazaki, Mitsuo Matsuda, and Iwao Yamaguchi. Physiological and pathological cardiac hypertrophy induce
different molecular phenotypes in the rat. Am J Physiol Regulatory Integrative Comp Physiol 281: R2029–R2036, 2001.—
Pressure overload, such as hypertension, to the heart causes
pathological cardiac hypertrophy, whereas chronic exercise
causes physiological cardiac hypertrophy, which is defined as
athletic heart. There are differences in cardiac properties between these two types of hypertrophy. We investigated whether
mRNA expression of various cardiovascular regulating factors
differs in rat hearts that are physiologically and pathologically
hypertrophied, because we hypothesized that these two types of
cardiac hypertrophy induce different molecular phenotypes. We
used the spontaneously hypertensive rat (SHR group; 19 wk
old) as a model of pathological hypertrophy and swim-trained
rats (trained group; 19 wk old, swim training for 15 wk) as a
model of physiological hypertrophy. We also used sedentary
Wistar-Kyoto rats as the control group (19 wk old). Left ventricular mass index for body weight was significantly higher in
SHR and trained groups than in the control group. Expression
of brain natriuretic peptide, angiotensin-converting enzyme,
and endothelin-1 mRNA in the heart was significantly higher in
the SHR group than in control and trained groups. Expression
of adrenomedullin mRNA in the heart was significantly lower
in the trained group than in control and SHR groups. Expression of ␤1-adrenergic receptor mRNA in the heart was significantly higher in SHR and trained groups than in the control
group. Expression of ␤1-adrenergic receptor kinase mRNA,
which inhibits ␤1-adrenergic receptor activity, in the heart was
markedly higher in the SHR group than in control and trained
groups. We demonstrated for the first time that the manner of
mRNA expression of various cardiovascular regulating factors
in the heart differs between physiological and pathological cardiac hypertrophy.
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METHODS
Animals and protocol. Experimental protocols were approved by the Committee on Animal Research at the University of Tsukuba. Male 4-wk-old Wistar-Kyoto (WKY) rats and
SHR were obtained from Charles River Japan (Yokohama,
Japan) and cared for according to the Guiding Principles for
the Care and Use of Animals, based on the Helsinki Declaration of 1964. The rats were maintained on a 12:12-h lightdark cycle and received food and water ad libitum. Eight
WKY rats were exercised by swimming for 5 days/wk
(trained group) in a tank of water at 30–32°C with a surface
area of 1,960 cm2 and a depth of 50 cm. The rats swam for 5
min/day for the first 2 days, and then the swim time was
increased gradually over the 2-wk period from 5 to 75 min/
day. Thereafter, the trained group continued swim training
for 13 wk. Therefore, the trained group received 15 wk of
swim training. Eight SHR (SHR group) and seven WKY rats
(control group) remained confined to their cages but were
handled daily. After swim training for 15 wk, the body weight
and hemodynamic parameters of the animals were measured, and the heart was removed, weighed, and frozen in
liquid nitrogen. Heart samples were stored at ⫺80°C for
determination of the expression levels of mRNA of various
cardiovascular regulating factors by reverse transcriptionpolymerase chain reaction (RT-PCR) analysis. Control rats
and SHR were killed at the same time point as the trained
rats: all were 19 wk old.
Hemodynamic measurement and tissue sampling. On the
day of the experiment, hemodynamic parameters were measured in anesthetized rats, as described previously (21, 29–
31, 38, 39), with minor modifications. The rats were anesthetized with thiobutabarbital (50 mg/kg body wt ip). After the
rats were fully sedated, arterial blood pressure and heart
rate (HR) were measured via a cannula in the carotid artery
with a pressure transducer (model SCK-590, Gould) connected to a polygraph system (amplifier: model AP-601G,
Nihon Kohden, Tokyo, Japan; tachometer: model AT-601G,
Nihon Kohden; thermal-pen recorder: model WT-687G, Nihon Kohden). Arterial blood pressure and HR were monitored and recorded continuously. Stroke volume (SV) was
measured by the thermodilution technique using a Cardiotherm 500 cardiac output computer (Columbus Instruments,
OH) equipped with a small animal interface (3, 18). The
thermistor microprobe catheter (Fr-1; Columbus Instruments) was inserted into the right carotid artery and advanced to the aortic arch. A catheter placed in the left jugular
vein was advanced to the right ventricle for rapid bolus
injection of 200 ␮l (plus catheter dead space) of cold saline.
The saline solution was injected with a Hamilton constantrate syringe to ensure rapid and repeatable injections of the
saline indicator solution. The cardiac output measurement
was repeated five times. Cardiac output was measured again
when the aortic blood temperature of the rat was ⱖ36°C, and
the arterial blood pressure returned to the baseline value.
Catheter placement was verified by postmortem examination. Body temperature of the rat was maintained at 37°C by
using a small animal warmer (model BWT-100, Bio Research
Center, Nagoya, Japan). SV was determined by dividing
cardiac output by the HR. After hemodynamic measurement,
the whole heart was rapidly excised and washed thoroughly
with cold saline to remove contaminating blood; then the left
ventricle was separated from the right ventricle and atria.
The left ventricle was weighed, frozen in liquid nitrogen, and
stored at ⫺80°C until extraction of total RNA.
Use of RT-PCR to determine levels of mRNA expression in
heart. Expression of BNP mRNA, ACE mRNA, ET-1 mRNA,
adrenomedullin mRNA, ␤1-adrenergic receptor mRNA, ␤1adrenergic receptor kinase mRNA, muscarinic M2 receptor
mRNA, and MHC mRNA in the left ventricle was analyzed
by RT-PCR. Expression of ␤-actin mRNA was determined as
an internal control. Semiquantitative RT-PCR was performed according to the method we described previously (9,
13, 20, 29, 38, 39).
Total tissue RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction with Isogen (Nippon
Gene, Toyama, Japan) according to the method described by
us previously (9, 13, 20, 29, 38, 39). Briefly, the tissue was
homogenized in Isogen (100 mg tissue/ml Isogen) with a
Polytron tissue homogenizer (model PT10SK/35, Kinematica,
Lucerne, Switzerland). The precipitated RNA was extracted
with chloroform, precipitated with isopropanol, and washed
with 75% (vol/vol) ethanol. The resulting RNA was resolved
in diethyl pyrocarbonate-treated water, treated with DNase I
(Takara, Shiga, Japan), and extracted again with Isogen to
eliminate the genomic DNA. The RNA concentration was
determined spectrophotometrically at 260 nm.
Total tissue RNA (10 ␮g) was primed with 0.05 ␮g of
oligo[d(pT)]12–18 and reverse transcribed by avian myeloblastosis virus RT using a first-strand cDNA synthesis kit (Life
Sciences). The reaction was performed at 43°C for 60 min.
The cDNA was diluted in a 1:10 ratio, and 1 ␮l was used for
PCR. Each PCR contained 10 mM Tris 䡠 HCl (pH 8.3), 50 mM
KCl, 1.5 mM MgCl2, dNTP at 200 ␮M each, gene-specific
primer at 0.5 ␮M each, and 0.025 U/␮l Taq polymerase
(Takara). The gene-specific primers were synthesized according to the published cDNA sequences for each of the following: BNP (15), ACE (14), ET-1 (33), adrenomedullin (32),
␤1-adrenergic receptor (19), ␤1-adrenergic receptor kinase
(2), muscarinic M2 receptor (8), MHC (17), and ␤-actin (23).
The sequences of the oligonucleotides were as follows: 5⬘-TA-
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uretic peptide (BNP) and a shift of isozyme from ␣- to
␤-myosin heavy chain (MHC) (10, 27, 29). It has been
reported that adrenomedullin, which is produced in
cardiac myocytes, inhibits hypertrophy of cardiac myocytes in vitro (5, 37). Therefore, it is considered that
various cardiovascular regulating factors, such as ANG
II, ET-1, BNP, adrenomedullin, ␤1-adrenergic pathway, and MHC, participate in the development of
pathological cardiac hypertrophy. Although the athletic heart exhibits physiological cardiac hypertrophy,
it is unknown whether the various cardiovascular regulating factors participate in the development of athletic heart induced by chronic exercise training.
Because there are differences in cardiac properties
between pathological and physiological cardiac hypertrophy, we hypothesized that the manner of mRNA
expression of various cardiovascular regulating factors
in the rat heart differs between these two types of
hypertrophy. Therefore, we investigated whether the
mRNA expression of various cardiovascular regulating
factors differs between physiological cardiac hypertrophy (athletic heart) and pathological cardiac hypertrophy. In the present study, we used the spontaneously
hypertensive rat (SHR; 19 wk old) as a model of pathological hypertrophy and swim-trained rats (trained
group; 19 wk old, swim training for 15 wk, 5 days/wk)
as a model of physiological hypertrophy. We also determined whether hemodynamic features differ in the
trained and SHR groups.
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CARDIOVASCULAR REGULATING FACTORS IN CARDIAC HYPERTROPHY
were scanned by CanoScan 600 (Canon, Tokyo, Japan), and
quantification was performed by a computer with MacBAS
software (Fuji Film, Tokyo, Japan) according to the method
described previously (9, 13, 20, 29, 38, 39). The cDNAs for the
verification of the semiquantitative PCR analysis were prepared from each gene PCR product of rat cDNA. Each PCR
product was purified, quantified, and used as a positivecontrol cDNA. We performed semiquantitative PCR analysis
to evaluate the expression levels of BNP mRNA, ACE mRNA,
ET-1 mRNA, adrenomedullin mRNA, ␤1-adrenergic receptor
mRNA, ␤1-adrenergic receptor kinase mRNA, muscarinic M2
receptor mRNA, MHC mRNA, and ␤-actin mRNA. To demonstrate that our semiquantitative PCR strategy was valid,
serial dilutions of each positive-control cDNA were amplified
by PCR and quantified by a scanner.
Statistical analysis. Values are means ⫾ SE. Statistical
analysis was carried out by analysis of variance followed by
Scheffé’s F test for multiple comparisons. P ⬍ 0.05 was
accepted as significant.
RESULTS
Hemodynamic parameters in control, trained, and
SHR groups. Body weight was significantly lower in
the trained group than in the control and SHR groups
(Table 1). Left ventricular mass index for body weight
was higher in the SHR and trained groups than in the
control group (Table 1). Resting HR was lower in the
trained group than in the control and SHR groups and
significantly higher in the SHR group than in the
control group (Table 1). Systolic and diastolic blood
pressure were significantly higher in the SHR group
than in the control and trained groups (Table 1). SV
was lower in the SHR group than in the control and
trained groups (Table 1). Pressure-rate product, which
is an index of cardiac workload, was higher in the SHR
group than in the control and trained groups (Table 1).
These results suggest that the heart of trained rats
exhibited physiological cardiac hypertrophy (athletic
heart), and the heart of SHR exhibited pathological
cardiac hypertrophy.
mRNA expression of cardiovascular regulating factors in heart. Expression of BNP mRNA, ACE mRNA,
and ET-1 mRNA in the heart was significantly higher
in the SHR group than in the control and trained
groups (Fig. 1). There was no significant difference
between the control and trained groups in expression
of BNP mRNA, ACE mRNA, and ET-1 mRNA (Fig. 1).
Expression of adrenomedullin mRNA in the heart was
Table 1. Body weight, tissue weight, and hemodynamic parameters in SHR, trained, and control rats
Body wt, g
Left ventricle wt/body wt, mg/g
Heart rate, beats/min
Blood pressure, mmHg
Systolic
Diastolic
Stroke volume, ␮l
Pressure-rate product, mmHg 䡠 beats 䡠 min⫺1
Control (n ⫽ 7)
SHR (n ⫽ 8)
Trained (n ⫽ 8)
391.7 ⫾ 5.8
2.34 ⫾ 0.19
354.5 ⫾ 16.1
379.0 ⫾ 6.9
2.76 ⫾ 0.11*
408.6 ⫾ 7.8*
338.8 ⫾ 4.4*†
2.63 ⫾ 0.14*
317.4 ⫾ 4.2*†
130.8 ⫾ 5.8
102.8 ⫾ 5.7
362.0 ⫾ 15.5
43,381.2 ⫾ 3,156.4
241.8 ⫾ 2.0*
183.1 ⫾ 1.8*
281.6 ⫾ 14.9*
87,990.5 ⫾ 2,060.7*
129.8 ⫾ 3.2†
103.1 ⫾ 2.5†
414.5 ⫾ 34.6†
38,170.5 ⫾ 1,201.2†
Values are means ⫾ SE. SHR, spontaneously hypertensive rat; trained, swim-trained (15 wk) rat. * P ⬍ 0.05 vs. corresponding value in
control rats. † P ⬍ 0.05 vs. corresponding value in SHR rats.
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ATCTGTCGCCGCTGGGAGG-3⬘ (sense) and 5⬘-GAGCTGGGGAAAGAAGAGCCG-3⬘ (antisense) for BNP, 5⬘-GTTCGTGGAGGAGTATGACCG-3⬘ (sense) and 5⬘-CCGTTGAGCTTGGCGATCTTG-3⬘ (antisense) for ACE, 5⬘-TCTTCTCTCTGCTGTTTGTG-3⬘ (sense) and 5⬘-TTAGTTTTCTTCCCTCCACC-3⬘
(antisense) for ET-1, 5⬘-GGTTCGCTCGCCGTTCTCG-3⬘
(sense) and 5⬘-GCCACCCGCACCTATAACCT-3⬘ (antisense)
for adrenomedullin, 5⬘-CGCTCACCAACCTCTTCATCATGTCC-3⬘ (sense) and 5⬘-CAGCACTTGGGGTCGTTGTAGCAGC-3⬘ (antisense) for ␤1-adrenergic receptor, 5⬘-AGATGGCCGACCTGGAGGC-3⬘ (sense) and 5⬘-GGAGTCAAAGATCTCCCG-3⬘ (antisense) for ␤1-adrenergic receptor kinase, 5⬘CACGAAACCTCTGACCTACCC-3⬘ (sense) and 5⬘-TCTGACCCGACGACCCAACTA-3⬘ (antisense) for muscarinic M2 receptor, 5⬘-GCAGACCATCAAGGACCT-3⬘ (sense) and 5⬘GTTGGCCTGTTCCTCCGCC-3⬘ (antisense) for MHC, 5⬘-GAAGATCCTGACCGAGCGTG-3⬘ (sense) and 5⬘-CGTACTCCTGCTTGCTGATCC-3⬘ (antisense) for ␤-actin. PCR was carried out using a PCR thermal cycler (model TP-3000, Takara)
according to the method described by us previously (9, 13, 20,
29, 38, 39). The cycle profile included denaturation for 15 s at
94°C, annealing for each suitable time at each suitable temperature, and extension for each suitable time at 72°C. The
annealing time and temperature were set as follows: 15 s at
70°C for BNP, 15 s at 69°C for ACE, 15 s at 54°C for ET-1,
15 s at 69°C for adrenomedullin, 30 s at 66°C for ␤1-adrenergic receptor, 15 s at 55°C for ␤1-adrenergic receptor kinase,
15 s at 71°C for muscarinic M2 receptor, 15 s at 63°C for
MHC, and 15 s at 72°C for ␤-actin. The extension time was
set as follows: 45 s for BNP, ACE, ET-1, ␤1-adrenergic receptor kinase, and MHC and 60 s for adrenomedullin, ␤1-adrenergic receptor, muscarinic M2 receptor, and ␤-actin. The
reaction cycles of PCR were performed in the range that
demonstrated a linear correlation between the amount of
cDNA and the yield of PCR products. After PCR in MHC,
distinction between ␣- and ␤-MHC was achieved by digestion
of 12.5 ␮l of the PCR mixture with 0.8 U of MseI in a standard
reaction buffer at 37°C for 4 h (29). This yielded fragments of
310 bp for ␣-MHC and 275 ⫹ 53 bp for ␤-MHC. The PCR
products were of the expected size, as shown by 1.5% agarose
gel electrophoresis for BNP, ACE, ET-1, adrenomedullin,
␤1-adrenergic receptor, ␤1-adrenergic receptor kinase, muscarinic M2 receptor, and ␤-actin and 2.0% agarose gel electrophoresis for MHC. In addition, the specificity of the amplified sequences was confirmed by restriction enzyme
analysis and DNA sequencing. The DNA sequence of each
amplicon was perfectly matched to each published sequence.
Semiquantitative analysis of PCR products. The amplified
PCR products were electrophoresed on 1.5% agarose gels,
stained with ethidium bromide, visualized by an ultraviolet
transilluminator, and photographed according to the method
described previously (9, 13, 20, 29, 38, 39). The photographs
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significantly lower in the trained group than in the
control and SHR groups (Fig. 2). Expression of ␤1adrenergic receptor mRNA in the heart was significantly higher in the SHR and trained groups than in
the control group (Fig. 3A). There was no significant
difference between the SHR and trained groups in
expression of ␤1-adrenergic receptor mRNA (Fig. 3A).
Expression of ␤1-adrenergic receptor kinase mRNA,
which inhibits ␤1-adrenergic receptor activity, in the
heart was markedly higher in the SHR group than in
the control and trained groups (Fig. 3B). There was no
significant difference between the control and trained
groups in expression of ␤1-adrenergic receptor kinase
(Fig. 3B). There were no significant differences among
the three groups in expression of muscarinic M2 receptor mRNA (Fig. 3C). Expression of ␣-MHC mRNA in
the heart was significantly higher in the trained group
than in the SHR group (Fig. 4A). There were no significant differences among the three groups in expression
of ␤-MHC mRNA (Fig. 4B).
DISCUSSION
We have demonstrated for the first time that the
manner of mRNA expression of various cardiovascular
regulating factors in the heart differed between physiological cardiac hypertrophy, which is induced by
chronic exercise, and pathological cardiac hypertrophy,
which is induced by hypertension. In the present study,
the hearts of SHR and trained rats developed cardiac
hypertrophy, as evidenced by an increase in left ventricular mass index for body weight. Trained rats received 15 wk of swim training, which caused enhancement of cardiac function, i.e., a decrease in resting HR
and pressure-rate product and an increase in SV. SHR
developed cardiac hypertrophy by hypertension and
showed a decline in cardiac function, i.e., increase in
resting HR and pressure-rate product and decrease in
SV. Therefore, these results suggest that trained rats
exhibited physiological cardiac hypertrophy (athletic
heart) and SHR exhibited pathological cardiac hypertrophy.
The present study revealed that expression of BNP
mRNA, ACE mRNA, and ET-1 mRNA in the heart was
significantly higher in the SHR group than in the
control and trained groups. It has been reported that
BNP is involved in pathological cardiac hypertrophy
(10). Furthermore, pathological cardiac hypertrophy is
partly induced by humoral cardiovascular regulating
factors such as ANG II (16, 40), which is converted
from ANG I by ACE, and ET-1 (11, 36). These humoral
cardiovascular regulating factors activate mitogen-activated protein kinase by the activation of GTP-binding
(Gq) protein (4, 41, 43), thereby resulting in cardiac
myocyte hypertrophy (6, 27). Furthermore, cardiac hypertrophy and contractile dysfunction have been reported in Gq-overexpressing mice (6). Therefore, it is
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Fig. 1. Expression of brain natriuretic peptide (BNP) mRNA (A), angiotensin-converting enzyme (ACE) mRNA (B),
and endothelin-1 (ET-1) mRNA (C) in the heart (left ventricle) of control rats (n ⫽ 7), spontaneously hypertensive
rats (SHR, n ⫽ 8), and trained rats (n ⫽ 8). Top: typical examples of RT-PCR analysis for levels of BNP, ACE, and
ET-1 mRNA. Bottom: results of statistical analysis of levels of expression of BNP, ACE, and ET-1 mRNA by a
densitometer. Expression of ␤-actin mRNA was determined as an internal control. Photos of PCR products were
scanned by a densitometer, and ratios of BNP, ACE, and ET-1 mRNA to ␤-actin mRNA were calculated. Thus
expression of BNP, ACE, and ET-1 mRNA was normalized by expression of ␤-actin mRNA. Values are means ⫾ SE.
Expression of BNP, ACE, and ET-1 mRNA in the heart was significantly higher in the SHR group than in control
and trained groups.
CARDIOVASCULAR REGULATING FACTORS IN CARDIAC HYPERTROPHY
considered that Gq overactivation induces heart failure. In the present study, the mRNA expression of
ACE and ET-1, which activate Gq protein, in the heart
was increased in the SHR group. Taken together, the
development of pathological cardiac hypertrophy in
SHR in the present study may be partly caused by
ANG II- and ET-1-induced activation of Gq protein. In
the present study, the expression of ACE and ET-1
mRNA in the heart was not altered by 15 wk of swim
training. Therefore, it is likely that activation of the
Gq-signaling pathway by ANG II or ET-1 may not
mainly participate in the development of physiological
cardiac hypertrophy. Furthermore, the expression of
BNP mRNA in the heart was not altered by exercise
training. Therefore, it is possible that physiological
cardiac hypertrophy (athletic heart) is induced by
other mechanisms.
Cardiac myocytes, as well as vascular endothelial
cells, produce adrenomedullin (5). It has been reported
that adrenomedullin inhibits hypertrophy of cardiac
myocytes in vitro (37). In the present study, the expression of adrenomedullin mRNA in the heart was significantly lower in the trained group than in the control
and SHR groups. Therefore, the decrease in expression
of adrenomedullin mRNA in the trained heart in this
study may have accelerated the development of physiological cardiac hypertrophy in the trained rats. It is
possible that a decrease in expression of myocardial
adrenomedullin partly participates in development of
the athletic heart.
The present study revealed that mRNA expression of
␤1-adrenergic receptor, which is a receptor in the signal transduction pathway in sympathetic nerve stimulation, in the heart was significantly higher in the
SHR and trained groups than in the control group.
However, mRNA expression of ␤1-adrenergic receptor
kinase, which inhibits activation of the signal transduction system downstream of the ␤1-adrenergic receptor by desensitization of the ␤1-adrenergic receptor, in
the heart was markedly higher in the SHR group than
in the control and trained groups. These findings suggest that in the heart of trained rats the signal transduction system downstream of ␤1-adrenergic receptor
was activated, whereas in SHR the signal transduction
system downstream of the ␤1-adrenergic receptor was
inactivated. Therefore, it is considered that the signal
transduction system of the ␤1-adrenergic receptor in
the heart differs between the athletic heart (physiological cardiac hypertrophy) and pathological cardiac hypertrophy. Sympathetic nervous system activity has
been implicated in development of cardiac hypertrophy
(27). An in vivo study also indicated that stimulation of
␤-adrenergic receptors leads to development of myocardial hypertrophy independent of hemodynamic effects
(42). Furthermore, Ji et al. (12) reported that ␤-adrenergic blockade prevented exercise training-induced
cardiac hypertrophy. On the basis of results from past
studies plus the present results, it is considered that
␤1-adrenergic system activity participates in development of the athletic heart (physiological cardiac hypertrophy). In the present study, there were no significant
differences among the three groups in mRNA expression of muscarinic M2 receptor, which binds acetylcholine released from parasympathetic nerve endings.
In the present study, expression of ␣-MHC mRNA in
the heart was significantly higher in the trained group
than in the SHR group. We also demonstrated that
expression of ␤-MHC mRNA in the heart was slightly
increased in the SHR group. It has been reported that
hypertension in rats results in a shift of myosin
isozymes from the predominant V1 to the V3 pattern
and that physical training by swimming in rats results
in an increase in the percentage of V1 myosin isozyme
in cardiac myosin (34). These observations are consistent with our findings. Therefore, these results suggest
that the myosin isozyme differs between the athletic
heart (physiological cardiac hypertrophy) and pathological cardiac hypertrophy. Furthermore, we suspect
that Ca2⫹-ATPase activity accelerates in the athletic
heart and is attenuated in pathological cardiac hypertrophy, because the V1 myosin isozyme accelerates
Ca2⫹-ATPase activity, whereas the V3 myosin isozyme
attenuates Ca2⫹-ATPase activity.
The mechanism underlying the differences in the
mRNAs measured between the athletic heart and
pathological cardiac hypertrophy remains to be elucidated. However, the following mechanism is possible.
Trained rats (athletic heart) induce tachycardia only
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Fig. 2. Expression of adrenomedullin mRNA in the heart (left ventricle) of control rats (n ⫽ 7), SHR (n ⫽ 8), and trained rats (n ⫽ 8).
Top: typical examples of RT-PCR analysis for levels of adrenomedullin mRNA. Bottom: result of statistical analysis of expression of
adrenomedullin mRNA by a densitometer. Expression of ␤-actin
mRNA was determined as an internal control. Photos of PCR products were scanned by a densitometer, and ratio of adrenomedullin
mRNA to ␤-actin mRNA was calculated. Thus expression of adrenomedullin mRNA was normalized by expression of ␤-actin
mRNA. Values are means ⫾ SE. Expression of adrenomedullin
mRNA in the heart was significantly lower in the trained group than
in control and SHR groups.
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Fig. 4. Expression of ␣- and ␤-myosin heavy chain
(␣-MHC and ␤-MHC) mRNA in the heart (left
ventricle) of control rats (n ⫽ 7), SHR (n ⫽ 8), and
trained rats (n ⫽ 8). Top: typical examples of
RT-PCR analysis for levels of ␣- and ␤-MHC
mRNA. Bottom: results of statistical analysis of
expression of ␣- and ␤-MHC mRNA by a densitometer. Expression of ␤-actin mRNA was determined as an internal control. Photos of PCR products were scanned by a densitometer, and ratios of
␣- and ␤-MHC mRNA to ␤-actin mRNA were
calculated. Thus ␣- and ␤-MHC mRNA expression
was normalized by ␤-actin mRNA expression. Values are means ⫾ SE. ␣-MHC mRNA expression in
the heart was significantly higher in the trained
group than in the SHR group.
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Fig. 3. Expression of ␤1-adrenergic receptor mRNA (A), ␤1-adrenergic receptor kinase mRNA (B), and muscarinic
M2 receptor mRNA (C) in the heart (left ventricle) of control rats (n ⫽ 7), SHR (n ⫽ 8), and trained rats (n ⫽ 8).
Top: typical examples of RT-PCR analysis for levels of ␤1-adrenergic receptor, ␤1-adrenergic receptor kinase, and
muscarinic M2 receptor mRNA. Bottom: results of statistical analysis of expression of ␤1-adrenergic receptor,
␤1-adrenergic receptor kinase, and muscarinic M2 receptor mRNA by a densitometer. Expression of ␤-actin mRNA
was determined as an internal control. Photos of PCR products were scanned by a densitometer, and ratios of
␤1-adrenergic receptor, ␤1-adrenergic receptor kinase, and muscarinic M2 receptor mRNA to ␤-actin mRNA were
calculated. Thus expression of ␤1-adrenergic receptor, ␤1-adrenergic receptor kinase, and muscarinic M2 receptor
mRNA were normalized by expression of ␤-actin mRNA. Values are means ⫾ SE. Expression of ␤1-adrenergic
receptor mRNA in the heart was significantly higher in SHR and trained groups than in the control group.
Expression of ␤1-adrenergic receptor kinase mRNA in the heart was markedly higher in the SHR group than in
control and trained groups.
CARDIOVASCULAR REGULATING FACTORS IN CARDIAC HYPERTROPHY
Perspectives
It is generally accepted that there are differences in
cardiac function between physiological and pathological cardiac hypertrophy (24, 26, 28). The present study
demonstrated a new molecular finding that the manner of mRNA expression of various cardiovascular regulating factors in the heart differed between physiological and pathological cardiac hypertrophy. These
findings showed that a different molecular mechanism
of formation of cardiac hypertrophy is possible between
these two types of cardiac hypertrophy. Further studies to precisely reveal molecular features of physiological and pathological cardiac hypertrophy are needed,
because these further studies will provide important
findings on molecular and cellular mechanism of formation of physiological and pathological cardiac hypertrophy. In this regard, it is of great interest and of
importance to study 1) whether the change in mRNA
expression between physiological and pathological cardiac hypertrophy contributes to a change in expression
of protein level or mature substance level in these
hearts, 2) whether there is a difference in the signaling
pathway mediated through these substances between
these two types of cardiac hypertrophy, and 3) how the
pharmacological action of these proteins or substances
on the cardiac myocytes differently alters in these two
types of cardiac hypertrophy. The present study demonstrated different molecular phenotypes between
physiological and pathological cardiac hypertrophy.
Therefore, the present study raised an important question, which should be solved by further studies: Is there
a difference in the molecular mechanism of formation
of cardiac hypertrophy between physiological and
pathological cardiac hypertrophy?
We thank Dr. Wendy Gray for editing the English language of our
manuscript.
This study was supported by grants-in-aid for scientific research
from the Ministry of Education, Science, Sports, and Culture of
Japan (00006781, 11557047, 12470147); and by a grant from the
Miyauchi Project of the Center for Tsukuba Advanced Research
Alliance at the University of Tsukuba.
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