Download Exercise Training Alters Left Ventricular Geometry

Exercise Training Alters Left Ventricular Geometry
and Attenuates Heart Failure in Dahl Salt-Sensitive
Hypertensive Rats
Masaaki Miyachi, Hiroki Yazawa, Mayuko Furukawa, Koji Tsuboi, Masafumi Ohtake,
Takao Nishizawa, Katsunori Hashimoto, Toyoharu Yokoi, Tetsuhito Kojima, Takashi Murate,
Mitsuhiro Yokota, Toyoaki Murohara, Yasuo Koike, Kohzo Nagata
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Abstract—The clinical efficacy of exercise training in individuals with heart failure is well established, but the mechanism
underlying such efficacy has remained unclear. An imbalance between cardiac hypertrophy and angiogenesis is
implicated in the transition to heart failure. We investigated the effects of exercise training on cardiac pathophysiology
in hypertensive rats. Dahl salt-sensitive rats fed a high-salt diet from 6 weeks of age were assigned to sedentary or
exercise (swimming)-trained groups at 9 weeks. Exercise training attenuated the development of heart failure and
increased survival, without affecting blood pressure, at 18 weeks. It also attenuated left ventricular concentricity without
a reduction in left ventricular mass or impairment of cardiac function. Interstitial fibrosis was increased and myocardial
capillary density was decreased in the heart of sedentary rats, and these effects were attenuated by exercise. Exercise
potentiated increases in the phosphorylation of Akt and mammalian target of rapamycin observed in the heart of
sedentary rats, whereas it inhibited the downregulation of proangiogenic gene expression apparent in these animals. The
abundance of the p110␣ isoform of phosphatidylinositol 3-kinase was decreased, whereas those of the p110␥ isoform
of phosphatidylinositol 3-kinase and the phosphorylation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase were increased, in the heart of sedentary rats, and all of these effects were prevented by exercise.
Thus, exercise training had a beneficial effect on cardiac remodeling and attenuated heart failure in hypertensive
rats, with these effects likely being attributable to the attenuation of left ventricular concentricity and restoration of
coronary angiogenesis through activation of phosphatidylinositol 3-kinase(p110␣)-Akt-mammalian target of rapamycin signaling. (Hypertension. 2009;53:00-00.)
Key Words: hypertension 䡲 sodium-dependent 䡲 heart failure 䡲 exercise 䡲 hypertrophy 䡲 rats
䡲 Dahl 䡲 coronary angiogenesis
.
H
the number of coronary capillaries and the size of cardiomyocytes, resulting in myocardial hypoxia, is thought to develop
during the progression of cardiac hypertrophy.6 Indeed, studies have indicated the existence of a relation among cardiac
angiogenesis, hypertrophy, and function.7,8 Attenuation of
coronary angiogenesis in the setting of load-induced cardiac
growth may, thus, play an important role in the development of
cardiac pathology, with the balance between cardiac growth and
coronary angiogenesis, rather than the extent of hypertrophy, per
se, being a key determinant of the transition from physiological
to pathological hypertrophy.2 Antiangiogenic activity of the
tumor suppressor protein p53 has been implicated recently in
the transition from cardiac hypertrophy to heart failure.9
The cardioprotective effects of exercise training are well
established. Studies have suggested that carefully applied
eart failure is a final common consequence of various
forms of heart disease and is a leading cause of mortality
worldwide. Cardiac hypertrophy associated with pathological
conditions such as hypertension, myocardial infarction, and
valvular heart disease has been thought to be an adaptive
response to increased external load, given that it can result in
normalization of the increase in wall stress induced by
mechanical overload. However, increased cardiac mass is
also associated with increased morbidity and mortality,1 with
sustained overload eventually leading to heart failure.
The serine-threonine protein kinase Akt is an important
mediator of phosphatidylinositol 3-kinase (PI3K) signaling
and regulates multiple cellular functions.2 PI3K-Akt signaling
is implicated in the regulation of cardiac growth, contractile
function, and coronary angiogenesis.3–5 A mismatch between
Received November 29, 2008; first decision December 19, 2008; revision accepted February 6, 2009.
From the Departments of Pathophysiology Laboratory Sciences (M.M., H.Y., K.T., M.O.) and Cardiology (T.N., T. Murohara), Nagoya University
Graduate School of Medicine, Nagoya, Japan; Department of Medical Technology (M.F., K.H., T.Y., T.K., T. Murate, Y.K., K.N.), Nagoya University
School of Health Sciences, Nagoya, Japan; Aichi-Gakuin University School of Dentistry (M.Y.), Nagoya, Japan.
Correspondence to Kohzo Nagata, Department of Medical Technology, Nagoya University School of Health Sciences, 1-1-20 Daikominami,
Higashi-ku, Nagoya 461-8673, Japan. E-mail [email protected]
© 2009 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.108.127290
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Hypertension
April 2009
programs of exercise in patients with heart failure are
generally safe and may improve exercise tolerance, vascular
endothelial function, central cardiac function, and overall
quality of life.10,11 Exercise training also appears to improve
survival in patients or animal models with heart failure.12–14
However, the mechanism underlying such efficacy has remained unclear.
We have now investigated the effects of exercise training
on cardiac growth, contractile function, and coronary angiogenesis, as well as on PI3K-Akt signaling in a rat model of
hypertension-induced heart failure. We hypothesized that
exercise training might alter left ventricular (LV) geometry
and induce myocardial angiogenesis and that such effects
might contribute to amelioration of heart failure.
Methods
Animals and Experimental Protocols
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Male inbred Dahl salt-sensitive (DS) rats fed an 8% NaCl diet after
6 weeks of age were assigned to sedentary (HF; n⫽16) or exercisetrained (Ex; n⫽8) groups at 9 weeks. Exercise training consisted of
swimming for 1 hour per day, 5 days per week, for 9 weeks. DS
rats maintained on a 0.3% NaCl diet after 6 weeks of age remain
normotensive, and such animals served as age-matched controls
(CNT group; n⫽8). At 18 weeks of age, all of the rats were
anesthetized by IP injection of ketamine (50 mg/kg of body
weight) and xylazine (10 mg/kg) and were subjected to echocardiographic and hemodynamic analyses. The heart was subsequently excised, and LV tissue was separated for analysis.
Extended details can be found in the online data supplement
(available at http://hyper.ahajournals.org).
Echocardiographic and Hemodynamic Analyses
Systolic blood pressure (SBP) and heart rate (HR) were measured
weekly in conscious animals by tail-cuff plethysmography (BP-98A;
Softron). At 18 weeks of age, rats were subjected to transthoracic
echocardiography, as described previously.15 Details of echocardiographic analysis are available in the online data supplement. After
echocardiography, cardiac catheterization was performed as described previously.16 Tracings of LV pressure and the ECG were
digitized to determine LV end-diastolic pressure.
Tissue Preparation
For details, please see the online data supplement.
Histology and Immunohistochemistry
The left ventricle was fixed in ice-cold 4% paraformaldehyde for 48 to
72 hours, embedded in paraffin, and processed for histology and
immunohistochemistry, as described.17 Sections were stained with
mouse monoclonal antibodies to the endothelial cell marker CD31
(diluted 1:100; Pharmingen) to determine the extent of coronary
capillary formation. Individual endothelial cells or clusters of endothelial cells, with or without a lumen, were regarded as capillaries.
Capillary density was expressed as the average number of capillaries per
square millimeter. The ratio of the number of coronary capillaries to that
of cardiomyocytes was also determined. All of the image analysis was
performed with National Institutes of Health Scion Image software.
Details are available in the online data supplement.
Quantitative RT-PCR Analysis
Total RNA was extracted from LV tissue and subjected to quantitative RT-PCR analysis, as described,18 with primers and TaqMan
probes specific for rat complementary DNAs encoding hypoxiainducible factor (HIF) 1␣ (5⬘-ACTGCACAGGCCACATTCATG-3⬘,
5⬘-CAGCACCAAGCACGTCATAGG-3⬘, and 5⬘-ACCAGCAGTAACCAGCCGCAGTGTG-3⬘ as the forward primer, reverse primer, and
TaqMan probe, respectively; GenBank accession No. NM㛭024359),
vascular endothelial growth factor (VEGF),19 and endothelial NO
synthase.20 Reagents for detection of human 18S rRNA (Applied
Biosystems) were used to quantify rat 18S rRNA as an internal standard.
Details are available in the online data supplement.
Immunoblot Analysis
Total protein was isolated from LV tissue and quantitated with the
Bradford reagent (Bio-Rad). Equal amounts of the total protein
fraction were subjected to SDS-PAGE, and the separated proteins
were transferred to a polyvinylidene difluoride membrane, as described previously.20 The membrane was incubated with a 1:1000
dilution of rabbit polyclonal antibodies to the p110␣ isoform of
PI3K, the p110␥ isoform of PI3K, Akt, Akt phosphorylated on
Ser473, mammalian target of rapamycin (mTOR), mTOR phosphorylated on Ser2448, p70 S6 kinase, p70 S6 kinase phosphorylated on
Thr389, p38 mitogen-activated protein kinase (MAPK), p38 MAPK
phosphorylated on Thr180 and Tyr182, extracellular signal-regulated
kinase (ERK) 1 and 2, or ERK1/2 phosphorylated on Thr202 and
Tyr204 (Cell Signaling Technology) and a dilution of a goat polyclonal antibody to GAPDH (Santa Cruz Biotechnology). It was then
exposed to a 1:1000 dilution of horseradish peroxidase– conjugated
goat antibodies to rabbit immunoglobulin G (Medical and Biological
Laboratories), after which immune complexes were detected and
quantified as described previously.20
Statistical Analysis
Data are presented as means⫾SEMs. Differences among groups
were assessed by 1-way factorial ANOVA; if a significant difference
was detected, intergroup comparisons were performed with Fisher’s
multiple-comparison test. The time courses of SBP and HR were
compared among groups by 2-way, repeated-measures ANOVA.
Survival rate was analyzed by the standard Kaplan–Meier method
with a log-rank test. A P value of ⬍0.05 was considered statistically
significant.
Results
LV Geometry, Cardiac Function, and Survival
SBP was significantly higher in the HF group than in the
CNT group at 7 weeks of age and thereafter (Figure 1A and
Table 1). It was slightly lower in the Ex group than in the HF
group from 9 weeks of age, when the animals in the Ex group
began exercise training, through 12 weeks of age. However,
there were no significant differences in SBP between the HF
and Ex groups from 6 through 18 weeks. HR was significantly higher in the HF group than in the CNT group at 7
weeks of age and thereafter (Figure 1B and Table 1). The Ex
group showed a significant reduction in HR from 10 to 12
weeks compared with the HF group. However, HR in the Ex
group began to increase again at 13 weeks and continued to
do so until 18 weeks, when it was almost the same as that in
the HF group. At 18 weeks of age, the ratio of LV weight:tibial
length, an index of cardiac hypertrophy, was increased by
58% in the HF group compared with the CNT group (Table
1). The ratio of lung weight:tibial length, an index of
pulmonary congestion, was also increased by 24% in the HF
group (Table 1). Exercise training did not affect the increase
in the LV weight:tibial length ratio, but it prevented that in
the lung weight:tibial length ratio. Although 9 (56%) of 16
rats died in the HF group during the experimental period (6
from heart failure and 3 before the development of heart
failure, probably from lethal arrhythmia), only 1 (13%) of 8
rats did so (from heart failure) in the Ex group (Table 1).
Kaplan–Meier analysis confirmed that the survival rate of
exercised rats was significantly greater than that of HF rats
(Figure 1C).
Miyachi et al
CNT
HF
Ex
200
**
150
100
6
9
12
Age (weeks)
HR (beats/min)
B 440
15
CNT
HF
Ex
380
9
12
Age (weeks)
15
18
C 100
Survival rate (%)
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6
†
80
60
40
20
0
*
CNT
HF
Ex
6
Exercise training
9
12
Age (weeks)
15
18
Figure 1. Time course of SBP (A) and HR (B), as well as
Kaplan–Meier plots of survival rate (C) in DS rats of the 3 experimental groups. Data are means⫾SEMs (n⫽8, 16, and 8 rats
initially for the CNT, HF, and Ex groups, respectively). *P⬍0.05
vs CNT group; †P⬍0.05 vs HF group.
Echocardiography revealed that the interventricular septum
thickness, LV posterior wall thickness, LV fractional shortening, LV mass, and the relative wall thickness (RWT) were
significantly greater and that LV end-diastolic dimension was
significantly smaller in the HF group than in the CNT group
(Table 2 and Figure 2A). Exercise training did not affect LV
Table 1. Physiological Parameters and Survival in DS Rats of
the 3 Experimental Groups at 18 Weeks of Age
Parameter
CNT (n⫽8)
HF (n⫽7)
Ex (n⫽7)
Body weight, g
410⫾11
367⫾4*
367⫾3*
SBP, mm Hg
130⫾5
213⫾3*
217⫾5*
HR, bpm
379⫾5
413⫾4*
408⫾1*†
Heart weight/TL, mg/mm
28.6⫾0.6
41.6⫾0.7*
41.6⫾1.1*
LV weight/TL, mg/mm
20.0⫾0.5
31.5⫾0.7*
31.3⫾0.8*
Lung weight/TL, mg/mm
36.1⫾0.8
44.7⫾1.9*
39.7⫾0.5†
4.1⫾0.2
8.3⫾1.2*
4.8⫾0.6†
100
44*
88†
LVEDP, mm Hg
Survival, %
Parameter
CNT (n⫽8)
HF (n⫽7)
Ex (n⫽7)
IVST, mm
1.46⫾0.05
2.67⫾0.08*
2.32⫾0.06*†
LVPWT, mm
1.48⫾0.04
2.52⫾0.07*
2.11⫾0.08*†
LVDd, mm
9.34⫾0.13
7.87⫾0.17*
8.65⫾0.14*†
LVFS, %
29.2⫾0.9
40.4⫾3.0*
32.0⫾1.5†
LV mass, mg
1005⫾50
1594⫾83*
1470⫾76*
RWT
0.31⫾0.01
0.66⫾0.02*
0.51⫾0.02*†
6.8⫾0.7
11.7⫾0.6*
9.8⫾0.6*†
Area of interstitial fibrosis, %
*†
*
400
360
18
Exercise training
420
3
Table 2. Echocardiographic and Morphological Parameters in
DS Rats of the 3 Experimental Groups at 18 Weeks of Age
Exercise training
TL indicates tibial length; LVEDP, LV end-diastolic pressure. Data are
means⫾SEMs unless otherwise specified.
*P⬍0.05 vs CNT group.
†P⬍0.05 vs HF group.
IVST indicates the thickness of the interventricular septum; LVPWT, the
thickness of LV posterior wall; LVDd, LV end-diastolic dimension; LVFS, LV
fractional shortening. Data are means⫾SEMs.
*P⬍0.05 vs CNT group.
†P⬍0.05 vs HF group.
mass, but it significantly attenuated the changes in interventricular septum thickness, LV posterior wall thickness, LV
fractional shortening, RWT, and LV end-diastolic dimension.
Hemodynamic analysis revealed that LV end-diastolic pressure was significantly increased in the HF group compared
with the CNT group and that exercise training attenuated the
load-induced increase in LVEDP (Table 1). Hemodynamic
overload resulted in a rightward and upward shift in the plot
of RWT versus LV mass, whereas exercise training resulted
in a downward shift with a slight shift to the left in this plot
(Figure 2B). These data indicate that exercise training attenuated LV concentricity without affecting LV mass or impairing cardiac function.
A
200 ms 2 mm
200 ms 2 mm
CNT
B
Relative wall thickness
SBP (mmHg)
A 250
Exercise and Heart Failure
200 ms 2 mm
HF
Ex
0.8
0.6
0.4
0.2
750
CNT
HF
Ex
1000
1250
1500
1750
2000
LV mass (mg)
Figure 2. Echocardiography of DS rats in the 3 experimental
groups at 18 weeks of age. A, Representative M-mode LV
echocardiograms of animals in the 3 groups. Scale bars represent 200 ms and 2 mm. B, Plot of RWT versus LV mass for animals in each group.
4
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April 2009
HF group compared with the CNT group, but it was not
affected further by exercise training (Figure 4E). The phosphorylation levels of both p38 MAPK and ERK1/2 were
significantly increased in the HF group compared with the
CNT group, and these effects were prevented by exercise
training (Figure 4F and 4G). Finally, the amounts of HIF-1␣,
VEGF, and endothelial NO synthase mRNAs in the left
ventricle were markedly reduced in the HF group compared
with the CNT group, and these changes were prevented by
exercise training (Figure 5).
Discussion
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Figure 3. Angiogenesis in the left ventricle of DS rats in the 3
experimental groups at 18 weeks of age. A, Representative
CD31 immunostaining for coronary capillaries. Scale bars,
50 ␮m. B and C, Capillary density (B) and the ratio of the number of capillaries to that of myocytes (C) in cross sections of the
LV myocardium. Data are means⫾SEMs (n⫽8, 7, and 7 for the
CNT, HF, and Ex groups, respectively). *P⬍0.05 vs CNT group;
†P⬍0.05 vs HF group.
Cardiac Fibrosis and Coronary Angiogenesis
Histological analysis revealed marked interstitial fibrosis in
the left ventricle of rats in the HF group compared with those
in the CNT group. This increase in cardiac fibrosis was
significantly reduced by exercise training (Table 2).
Immunostaining of the myocardium with antibodies to
CD31 to detect capillary endothelial cells revealed that
capillary density was decreased in the HF group as a result of
the pronounced cardiac hypertrophy and that exercise training
restored capillary density to the level apparent in the CNT
group despite the remaining cardiac hypertrophy (Figure 3A
and 3B). The ratio of the number of coronary capillaries to
that of cardiomyocytes was slightly but significantly increased in the HF group compared with the CNT group, and
exercise training induced an additional increase in this ratio
(Figure 3C).
Activation of Proangiogenic Signaling
The myocardial abundance of the p110␣ isoform of PI3K was
markedly reduced, whereas that of the p110␥ isoform of PI3K
was significantly increased, in the HF group compared with
the CNT group. These changes in PI3K isoform expression
were prevented by exercise training (Figure 4A and 4B). The
ratio of the amount of the phospho-Ser473 form of Akt to that
of total Akt was significantly increased in the HF group
compared with the CNT group, and exercise training resulted
in a further increase in this ratio (Figure 4C). The phosphorylation of mTOR on Ser2448 was also increased in the HF
group compared with the CNT group and was increased
further by exercise training (Figure 4D). The phosphorylation
of p70 S6 kinase on Thr389 was significantly increased in the
We have shown that exercise training altered LV geometry,
restored coronary capillary growth, and attenuated the development of decompensated heart failure in hypertensive DS
rats. The beneficial effects of exercise training in this model
are likely attributable, at least in part, to amelioration of the
imbalance between cardiac growth and angiogenesis through
activation of the PI3K(p110 ␣ )-Akt-mTOR signaling
pathway.
Exercise training did not significantly affect SBP during
the experimental period, although the increase in SBP induced by the high-salt diet was slightly attenuated for several
weeks after the initiation of exercise, consistent with previous
observations in spontaneously hypertensive rats and heart
failure rats.13,14,21 Exercise training increases venous return,
leading to an increase in cardiac output, contributing to the
preserved hypertension, especially in the setting of high salt
intake. The responses of SBP to exercise, thus, likely depend
on the form and the intensity and duration of exercise or on
the extent of chronic volume load. HR was significantly
lowered by exercise training during the initial few weeks.
Exercise training increases baroreflex and overall vagal
activity, and a slow HR at rest is in general associated with
greater longevity.22 Indeed, exercise training inhibited the
development of heart failure and increased survival rate in the
present model of hypertension, consistent with the results of
previous studies showing that exercise training induces bradycardia and improves survival in hypertensive rat models.13,14,21,23 The return to an increasing HR at 13 weeks in the
Ex group in the present study might be attributable to the
progression of cardiac pathophysiology resulting from persistent severe hypertension.
The adaptations induced by exercise training, including
physiological hypertrophy, may be regarded as compensatory
responses to a chronic volume load. Patients with essential
hypertension manifest 4 different patterns of LV geometry in
terms of LV mass and RWT.24 Mortality and the frequency of
cardiovascular events are highest in patients with concentric
hypertrophy and intermediate in those with eccentric hypertrophy.24 –26 In the present study, exercise training attenuated
LV concentricity without affecting LV mass or impairing
cardiac function. It thereby attenuated heart failure and
increased survival in hypertensive DS rats, consistent with
previous observations with other animal models of hypertension.13,14 The physiological form of cardiac hypertrophy is an
adaptive response to long-term exercise training, whereas the
pathological form is often a maladaptive response to provocative stimuli, such as hypertension and aortic stenosis.3 The
Miyachi et al
B
PI3K
(p110α)
PI3K
(p110γ)
CNT
HF
p-mTOR
(Ser 2488)
Akt
CNT
Ex
1.5
HF
Ex
1.0
*
0.5
CNT
HF
*
2.0
†
1.5
1.0
0.5
0
Ex
HF
CNT
HF
*†
4.0
3.0
*
2.0
1.0
0
Ex
CNT
HF
F
G
p-p70S6K
(Thr 389)
p-p38 MAPK
(Thr 180 /Tyr 182)
p-ERK 1/2
(Thr 202 /Tyr 204)
HF
Ex
1.0
0.5
CNT
HF
*
1.5
†
1.0
0.5
0
*
2.0
1.0
CNT
HF
Ex
HF
Ex
2.5
2.0
Ex
*†
3.0
0
Ex
CNT
Ex
Relative phoshorylation
Relative phoshorylation
Relative phoshorylation
*
*
1.5
0
HF
2.5
2.0
Ex
ERK 1/2
CNT
2.5
CNT
HF
2.0
1.5
†
1.0
0.5
0
Ex
*
CNT
HF
Ex
Figure 4. Abundance of p110␣ and p110␥ isoforms of PI3K, as well as phosphorylation of Akt, mTOR, p70 S6 kinase, p38 MAPK, and
ERK, in the left ventricle of DS rats in the 3 experimental groups at 18 weeks of age. Representative immunoblots and either the ratio
of the amounts of p110␣ (A) or p110␥ (B) to that of GAPDH or the ratio of phosphorylated (p-) to total forms of Akt (C), mTOR (D), p70
S6 kinase (p70S6K; E), p38 MAPK (F), or ERK1/2 (G) are shown. Quantitative data are expressed relative to the corresponding value for
the CNT group and are means⫾SEMs (n⫽8, 7, and 7 for the CNT, HF, and Ex groups, respectively). *P⬍0.05 vs CNT group; †P⬍0.05
vs HF group.
physiological hypertrophy apparent in the heart of athletes
manifests as eccentric hypertrophy.
The progression to heart failure is typically associated with
increased fibrosis and disruption of normal cellular organization in the LV myocardium.27,28 Exercise training reduced the
extent of interstitial fibrosis in the left ventricle of hypertensive DS rats, consistent with the notion that physiological
hypertrophy is not associated with interstitial fibrosis.2,3 The
attenuation of LV concentricity observed in the Ex group,
thus, likely reflects a compensatory morphological response
to maintain cardiac function.
The balance between cardiac growth and coronary angiogenesis is a key determinant of cardiac function, with distur-
†
1.0
*
0.5
0
CNT
HF
Ex
C
1.5
1.0
†
*
0.5
0
CNT
HF
Ex
eNOS mRNA/18S rRNA
B
1.5
VEGF mRNA/18S rRNA
mRNA/18S rRNA
A
HIF-1
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p38 MAPK
CNT
HF
4.0
E
p70S6K
CNT
Ex
5.0
Relative phoshorylation
†
5
mTOR
CNT
2.5
Relative abundance
Relative abundance
D
p-Akt
(Ser 473)
GAPDH
GAPDH
0
C
Relative phoshorylation
A
Exercise and Heart Failure
bance of this balance being implicated in the transition from
adaptive hypertrophy to heart failure.29 Physiological cardiac
hypertrophy is associated with a normal or increased number
of myocardial capillaries, whereas pathological hypertrophy
is associated with a reduction in capillary density.30 Indeed,
myocardial capillary density is reduced in patients with heart
disorders, eg, aortic stenosis, dilated cardiomyopathy, or
ischemic cardiomyopathy.31,32 In the present study, exercise
training reduced the extent of cardiac interstitial fibrosis and
restored coronary capillary density, as well as attenuated LV
concentricity in hypertensive DS rats. In addition, exercise
training induced a further increase in the ratio of the number
of coronary capillaries to that of cardiomyocytes in these
1.5
†
1.0
*
0.5
0
CNT
HF
Ex
Figure 5. Expression of proangiogenic
genes in the left ventricle of DS rats in the 3
experimental groups at 18 weeks of age.
The abundance of HIF-1␣ (A), VEGF (B),
and endothelial NO synthase (eNOS; C)
mRNAs was determined by RT-PCR analysis, normalized by that of 18S rRNA, and
then expressed relative to the normalized
value for the CNT group. Data are
means⫾SEMs (n⫽8, 7, and 7 for the CNT,
HF, and Ex groups, respectively). *P⬍0.05
vs CNT group; †P⬍0.05 vs HF group.
6
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April 2009
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animals. These data are consistent with those of previous
studies showing that capillary density was increased in the
hypertrophic left ventricle by exercise training.21,33 The
exercise-induced normalization of the imbalance between
cardiac growth and coronary angiogenesis may, thus, have
contributed to the preservation of cardiac function and the
consequent improvement in the survival of hypertensive DS rats.
Akt is activated by various extracellular stimuli in a
PI3K-dependent manner and regulates multiple aspects of
cellular functions, including survival, growth, and metabolism.2 Akt is required for physiological cardiac growth.3
However, in Akt transgenic mice, long-term Akt activation
results in excessive cardiac hypertrophy associated with
pathological remodeling and loss of contractile function.29
Akt is activated by the p110␣ isoform of PI3K in the
induction of physiological hypertrophy, but it is also activated
by the p110␥ isoform of PI3K in response to various agonists
of G protein– coupled receptors, eg, endothelin 1, in the
induction of pathological hypertrophy.4 It is, therefore, unlikely that PI3K-Akt signaling is the sole determinant of
physiological versus pathological hypertrophy. It is possible,
however, that the intensity or duration of signaling mediated
by p110␣ differs from that mediated by p110␥ and that such
a difference contributes to the induction of physiological
versus pathological hypertrophy.34 In the present study, the
abundance of p110␣ was markedly reduced, whereas that of
p110␥ was significantly increased, in the left ventricle of HF
rats. In addition, the increase in the phosphorylation levels of
Akt and mTOR in HF rats was not accompanied by an
increase in myocardial capillary density. Furthermore, the
activation of p38 MAPK and ERK apparent in the HF group
is indicative of the development of pathological hypertrophy.5,34 In contrast, exercise training inhibited the isoform
shift of PI3K, as well as the activation of p38 MAPK and
ERK, with these effects likely underlying both the prevention
of the decrease in myocardial capillary density and the further
increase in the extents of Akt and mTOR phosphorylation.
Exercise activates the insulin-like growth factor
1-PI3K(p110␣) pathway, and it has been shown that
PI3K(p110␣) plays a critical role in the induction of cardiac
growth induced by exercise training.5 The p110␣ isoform of
PI3K has also been suggested to inhibit signaling at the level
of G protein– coupled receptors or G proteins.4 It is, thus,
likely that physiological stimuli (eg, exercise training) cannot
only switch on the IGF1-PI3K(p110␣) pathway but also
switch off the G protein– coupled receptor agonistPI3K(p110␥) pathway activated by pathological stimuli (eg,
pressure overload).4 Together, these observations suggest that
exercise training reduced the imbalance between cardiac
growth and angiogenesis through activation of the
PI3K(p110␣)-Akt-mTOR pathway.
VEGF is a central regulator of angiogenesis. Akt-induced
expression of VEGF is mediated by activation of mTOR and
consequent upregulation of HIF-1.35 The mTOR-dependent
expression of VEGF is upregulated during the physiological
phase of cardiac growth but is downregulated during the
pathological phase of cardiac hypertrophy.29 In the present
study, the increase in the phosphorylation of mTOR was
accompanied by phosphorylation of p70 S6 kinase in the left
ventricle of HF rats. However, the abundance of HIF-1␣,
VEGF, and endothelial NO synthase mRNAs was reduced in
the left ventricle of these animals. Exercise-induced stimulation of Akt-mTOR signaling was associated with restoration
of the expression of the genes for these proangiogenic
proteins. The phosphorylation of p70 S6 kinase was not
affected by exercise training. These data suggest that shortterm Akt activation promotes coronary angiogenesis in a
manner dependent on mTOR and that long-term activation of
Akt-mTOR signaling results in its uncoupling from stimulation of the expression of proangiogenic proteins, leading to
impaired coronary angiogenesis and excessive growth of the
heart.29 The underlying mechanisms responsible for exerciseinduced restoration of coronary angiogenesis remain to be
determined. It is possible that the increase in coronary blood
flow and sheer stress induced by exercise training improves
vascular endothelial function or that the antioxidant or antiinflammatory effects of exercise training result in inhibition
of myocardial fibrosis and stimulation of coronary capillary
formation.
With regard to limitations of the present study, we did not
assess whether the intensity of exercise used was the most
appropriate in this model of hypertension. The duration of
exercise training was based on previous observations,21 but
the efficacy of shorter durations of exercise has also been
demonstrated.13,14 Excessive long-term exercise was shown
to promote cardiac fibrosis and to have deleterious effects on
cardiac remodeling28,36 or to lead to bronchial congestion
resulting from increased left atrial pressure,37 possibly accelerating the progression to heart failure. Recently, the Heart
Failure and a Controlled Trial Investigating Outcomes of
Exercise Training Trial demonstrated that an exercise training
program in patients with heart failure is safe and may
contribute to the reduction of clinical events but does not
improve the short-term survival.38 Exercise regimens must,
therefore, be carefully calibrated for clinical application to
heart failure.39
Perspectives
We have shown that exercise training had a beneficial effect
on cardiac remodeling and attenuated heart failure in hypertensive DS rats. It promoted coronary angiogenesis mediated
by PI3K(p110␣)-Akt-mTOR signaling and attenuated LV
concentricity and inhibited myocardial fibrosis, leading to the
preservation of cardiac function and improved survival.
Given that the balance between cardiac growth and angiogenesis is a key determinant of cardiac function, it may be
advantageous to stimulate angiogenesis as part of a general
strategy to prevent or reverse heart failure. It may be possible
to treat hypertension and heart failure with a combination of
antihypertrophic (antihypertensive drugs) and proangiogenic
(exercise training) protocols, with the combined therapy
being more effective than either treatment alone.
Sources of Funding
This work was supported by unrestricted research grants from
Takeda Pharmaceutical Co Ltd (Osaka, Japan), Chugai Pharmaceutical Co Ltd (Tokyo, Japan), and Kyowa Hakko Kogyo Co, Ltd
(Tokyo, Japan), and by Management Expenses Grants from the
government to Nagoya University.
Miyachi et al
Acknowledgments
We thank Ayako Fukata, Yuriko Kato, and Yurie Kasai for technical
assistance.
Disclosures
None.
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Exercise Training Alters Left Ventricular Geometry and Attenuates Heart Failure in Dahl
Salt-Sensitive Hypertensive Rats
Masaaki Miyachi, Hiroki Yazawa, Mayuko Furukawa, Koji Tsuboi, Masafumi Ohtake, Takao
Nishizawa, Katsunori Hashimoto, Toyoharu Yokoi, Tetsuhito Kojima, Takashi Murate,
Mitsuhiro Yokota, Toyoaki Murohara, Yasuo Koike and Kohzo Nagata
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Hypertension. published online March 2, 2009;
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Online supplement
EXERCISE TRAINING ALTERS LEFT VENTRICULAR GEOMETRY AND
ATTENUATES HEART FAILURE IN DAHL SALT-SENSITIVE HYPERTENSIVE
RATS
Short title: Exercise and heart failure
Masaaki Miyachi,* Hiroki Yazawa,* Mayuko Furukawa,† Koji Tsuboi,* Masafumi Ohtake,*
Takao Nishizawa,‡ Katsunori Hashimoto,† Toyoharu Yokoi,† Tetsuhito Kojima,† Takashi
Murate,† Mitsuhiro Yokota,§ Toyoaki Murohara,‡ Yasuo Koike,† and Kohzo Nagata†
*Department of Pathophysiology Laboratory Sciences and ‡Department of Cardiology,
Nagoya University Graduate School of Medicine, Nagoya, Japan; †Department of Medical
Technology, Nagoya University School of Health Sciences, Nagoya, Japan; §Aichi-Gakuin
University School of Dentistry, Nagoya, Japan
Address correspondence to: Kohzo Nagata, MD, PhD, Department of Medical Technology,
Nagoya University School of Health Sciences, 1-1-20 Daikominami, Higashi-ku, Nagoya
461-8673, Japan.
Tel./Fax: +81-52-719-1546. E-mail: [email protected]
1
I. Expanded Materials and Methods
Animals and Experimental Protocols
Male inbred Dahl salt-sensitive (DS) rats were obtained from Japan SLC, Inc. (Hamamatsu,
Japan) and were handled in accordance with the guidelines of Nagoya University Graduate
School of Medicine as well as with the Guide for the Care and Use of Laboratory Animals
(NIH publication no. 85-23, revised 1996). Weaning rats were fed laboratory chow containing
0.3% NaCl until 6 weeks of age. DS rats fed an 8% NaCl diet after 6 weeks manifest
hypertension-induced compensated cardiac hypertrophy at 11 weeks and a distinct stage of
decompensated heart failure at 18 weeks. Exercise training consisted of swimming for 1 h per
day on 5 days per week for 9 weeks. The rats swam in a pool (50 × 50 × 70 cm) filled with
water to a depth of 50 cm. The water temperature was maintained at 36° ± 1°C. Swimming is
thought to be a natural behavior of rodents, and in addition, it is less stressful than, and avoids
electric shock and foot injury associated with, other forced exercise protocols. Both the diets
and tap water were provided ad libitum throughout the experimental period.
Echocardiographic Analysis
M-mode echocardiography was performed with a 12.5-MHz transducer (Aplio SSA-700A;
Toshiba, Tochigi, Japan). LV end-diastolic (LVDd) and end-systolic (LVDs) dimensions and
the thickness of the interventricular septum (IVST) and LV posterior wall (LVPWT) were
measured, and LV fractional shortening (LVFS), relative wall thickness (RWT), and LV mass
were calculated as follows: LVFS (%) = [(LVDd – LVDs)/LVDd] × 100; RWT = (IVST +
LVPWT)/LVDd; LV mass (g) = ({1.04 × [(IVST + LVDd + LVPWT)3 – (LVDd)3]} × 0.8) +
0.14.1
Tissue Preparation
The left ventricle was cut transversely into four portions comprising the apex, two middle
rings (each 3 mm in thickness), and the base. For histology, one of the middle-ring specimens
was fixed in ice-cold 4% paraformaldehyde for 48 to 72 h and then embedded in paraffin; for
preparation of frozen sections, the other middle-ring specimen was rapidly frozen in
isopentane cooled with liquid nitrogen and was then embedded in OCT compound within a
cryomold. The apex specimen was rapidly frozen in liquid nitrogen and stored at –80°C until
analysis.
Histology and Immunohistochemistry
Sections were cut from paraffin blocks at a thickness of 3 μm and were stained with Azan
Mallory solution for evaluation of fibrosis, as described previously.2 To determine the extent
of coronary capillary formation, we performed immunostaining for the endothelial cell
marker CD31 with frozen sections (thickness, 5 μm) that had been fixed with acetone.
Endogenous peroxidase activity was blocked by exposure of the sections to methanol
containing 0.3% hydrogen peroxide. Sections were incubated at 4°C first overnight with
mouse monoclonal antibodies to CD31 (diluted 1:100; Pharmingen, San Diego, CA) and then
for 30 min with Histofine Simple Stain Rat MAX PO (Nichirei Biosciences, Tokyo, Japan).
Immune complexes were visualized with diaminobenzidine and hydrogen peroxide, and the
sections were counterstained with hematoxylin. Capillaries and surrounding cardiomyocytes
were counted in five different microscopic fields (×400) of each section.
Quantitative RT-PCR Analysis
Total RNA was extracted from LV tissue and treated with DNase with the use of an SV Total
RNA Isolation System (Promega, Madison, WI). Complementary DNA (cDNA) was
synthesized by reverse transcription (RT) from 2 µg of total RNA with the use of random
primers (Invitrogen, Carlsbad, CA) and MuLV Reverse Transcriptase (Applied Biosystems,
Foster City, CA). Quantitative polymerase chain reaction (PCR) analysis was performed with
a Prism 7700 Sequence Detector (Perkin-Elmer, Waltham, MA), with primers and TaqMan
probes specific for rat cDNAs encoding hypoxia-inducible factor (HIF)-1α
2
(5′-ACTGCACAGGCCACATTCATG-3′, 5′-CAGCACCAAGCACGTCATAGG- 3′, and
5′-ACCAGCAGTAACCAGCCGCAGTGTG-3′ as the forward primer, reverse primer, and
TaqMan probe, respectively; GenBank accession no. NM_024359), vascular endothelial
growth factor (VEGF),3 and endothelial nitric oxide synthase (eNOS).4 Reagents for
detection of human 18S rRNA (Applied Biosystems) were used to quantify rat 18S rRNA as
an internal standard.
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3