Download Upregulation of Heat Shock Transcription Factor 1 Plays a Critical

Upregulation of Heat Shock Transcription Factor 1 Plays a
Critical Role in Adaptive Cardiac Hypertrophy
Masaya Sakamoto,* Tohru Minamino, Haruhiro Toko,* Yosuke Kayama,* Yunzeng Zou,
Masanori Sano, Eiichi Takaki, Teruhiko Aoyagi, Katsuyoshi Tojo, Naoko Tajima, Akira Nakai,
Hiroyuki Aburatani, Issei Komuro
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Abstract—Exercise-induced cardiac hypertrophy has been reported to have better prognosis than pressure overloadinduced cardiac hypertrophy. Cardiac hypertrophy induced by exercise was associated with less cardiac fibrosis and
better systolic function, suggesting that the adaptive mechanisms may exist in exercise-induced hypertrophy. Here, we
showed a critical role of heat shock transcription factor 1 (HSF1), an important transcription factor for heat shock
proteins, in the adaptive mechanism of cardiac hypertrophy. We examined expression of 8800 genes in the heart of
exercise-induced hypertrophy model using DNA chip technique and compared with pressure overload–induced
hypertrophy. Expression of HSF1 and its target molecule heat shock proteins was significantly upregulated in the heart
by exercise but not by chronic pressure overload. Constitutive activation of HSF1 in the heart significantly ameliorated
death of cardiomyocytes and cardiac fibrosis and thereby prevented cardiac dysfunction as well as hypertrophy induced
by chronic pressure overload. Conversely, decreased activity of HSF1 in the heart promoted cardiac dysfunction in
response to exercise, a load that normally leads to adaptive hypertrophy with preserved systolic function. Likewise,
cardiac function was significantly impaired from the early phase of pressure overload, when HSF1 activation was
inhibited. These results suggest that HSF1 plays a critical role in the transition between adaptive and maladaptive
hypertrophy. (Circ Res. 2006;99:1411-1418.)
Key Words: pressure overload 䡲 exercise 䡲 heart failure
C
ardiac hypertrophy is an adaptive response to increased
wall stress. At the beginning, cardiac hypertrophy has
beneficial effects to maintain cardiac output by reducing wall
stress; however, long-term stresses induce systolic dysfunction, leading to heart failure.1 Clinical studies have demonstrated that cardiac hypertrophy is not only a cause of
congestive heart failure but also an independent risk factor for
myocardial infarction, arrhythmia, and sudden death.2 Therefore, it is very important to elucidate the as yet uncharacterized mechanisms underlying the transition from cardiac
hypertrophy to heart failure and to develop potential therapeutic strategies for this condition.
A metaanalysis of the hearts of athletes has demonstrated
that the endurance-trained heart shows an increase in both left
ventricular (LV) diameter and LV wall thickness without
impairment of systolic and diastolic function.3 Because
exercise-induced cardiac hypertrophy, the so called “sports
heart,” has been reported not to progress to heart failure,4,5 it
is thought to be “adaptive hypertrophy.” On the other hand,
pressure overload initially induces adaptive hypertrophy, but
it leads to “maladaptive hypertrophy” in the chronic phase,
resulting in heart failure.6 Although there have been reports
demonstrating different expression patterns of some genes
between these 2 forms of cardiac hypertrophy,7,8 the precise
mechanism of different prognosis remains unclear.
In the present study, we examined expression of 8800
genes in the hearts of 2 hypertrophy rat models using DNA
chip. In the DNA chip analysis, we found that the heat shock
protein (Hsp) genes such as Hsp70 and Hsp27 were markedly
upregulated in the heart of the exercise model but not in that
of the pressure-overload model. Heat shock transcription
factor 1 (HSF1), which regulates Hsps gene expression,9 –11
was activated only in the heart of the exercise model.
Overexpression of activated HSF1 in the heart prevented
cardiomyocyte death and cardiac fibrosis in response to
sustained pressure overload and thereby preserved cardiac
Original received March 31, 2006; resubmission received September 19, 2006; revised resubmission received October 28, 2006; accepted October 31,
2006.
From the Department of Cardiovascular Science and Medicine (M. Sakamoto, T.M., H.T., Y.K., Y.Z., M. Sano, I.K.), Chiba University Graduate
School of Medicine; Department of Biochemistry and Molecular Biology (E.T., A.N.), Yamaguchi University Graduate School of Medicine, Ube; Japan
Red Cross Medical Center (T.A.), Tokyo; Division of Diabetes, Metabolism and Endocrinology (M. Sakamoto, K.T., N.T.), Department of Internal
Medicine, Jikei University School of Medicine, Tokyo; and Genome Science Division (H.A.), Research Center for Advanced Science and Technology,
University of Tokyo, Japan.
*These authors contributed equally to this work.
Correspondence to Issei Komuro, MD, PhD, Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine,
1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail [email protected]
© 2006 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000252345.80198.97
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December 8/22, 2006
function. Conversely, decreased expression of HSF1 in the
heart impaired the adaptive response to exercise or acute
pressure overload and thus caused cardiac dysfunction, suggesting a protective role of HSF1 in cardiac physiology.
Materials and Methods
Rat Models
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All protocols were approved by the Institutional Animal Care and
Use Committee of Chiba University. Male Wistar rats obtained from
Nippon Bio-Supply Center (Tokyo, Japan) were divided into 3
groups: sham-operated model, exercise-induced hypertrophy model,
and pressure overload–induced hypertrophy model (n⫽8, each
group). Five-week-old male rats were individually housed and
voluntary exercised in a specially manufactured cage equipped with
a controlled running wheel and a distance counter as described
previously.12 In this cage, there were 2 rooms (a room for wheel
running and another for rest), and therefore rats could run or rest ad
libitum. Pressure overload was produced by constriction of abdominal aorta as described previously.13 Briefly, 8-week-old male rats
were anesthetized with an intraperitoneal injection of a cocktail of
ketamine HCl (100 mg/kg) and xylazine (5 mg/kg). The abdominal
aorta was constricted above the right renal artery by 4-0 silk suture
tied around both the aorta and a blunted 22-gauge needle, which was
then pulled out.
lary profiles and nuclei (n⫽100, each group).18 To determine the
degree of collagen fiber accumulation, we selected 5 fields at random
and calculated the ratio of Masson-stained fibrosis area to total
myocardium area with the software “NIH Image” (Bethesda, Md) for
image analysis as described previously.19 Cardiomyocyte death was
detected in situ by terminal deoxyribonucleotide transferase–mediated dUTP nick-end labeling (TUNEL) method using Cardio TACS
(Trevigen Inc, Gaithersburg, Md), in paraffin-embedded heart tissue
sections.
Ribonuclease Protection Assay and Northern and
Western Blot Analysis
Total RNA (20 ␮g) was separated on a 1.0% agarose/formaldehyde
gel and was hybridized with the cDNA fragments of Hsp70, Hsp27,
and HSF1 genes. Ribonuclease protection assay (RiboQuant, Pharmingen, Franklin Lakes, NJ) was performed according to the instructions of the manufacturer. Western blotting was performed as
previously described.15 Antibody for inducible Hsp70 was purchased
from Stressgen Biotechnologies (Victoria, Canada) and antibody for
HSF1 was from Santa Cruz Biotechnology (Santa Cruz, Calif).
Gel Mobility-Shift Assay
DNA-binding activity of HSF1 was examined using a selfcomplementary oligonucleotide containing the heat shock element
(5⬘-CTAGAAGCTTCTAGAAGCTTCTAG-3⬘) as a probe.15
DNA Chip Analysis
ECG Analysis
Total RNA (5 ␮g) was extracted from LV of each model by the
lithium/urea method and was used to synthesize biotin-labeled
cRNA, which was then hybridized to high-density oligonucleotide
array (GeneChip U34A array; Affymetrix, Santa Clara, Calif)
according to the previously published protocol.14 Arrays contain
probe sets for approximately 8800 genes and expressed-sequence
tags, which were selected from Build no. 34 of the UniGene
Database (created from GenBank 107/dbEST, November 18, 1998).
The GeneChip 3.3 software (Affymetrix) was used to calculate the
average difference for each probe on the array, which was shown as
an intensity value of gene expression defined by Affymetrix using
their algorithm. The average difference has been shown to quantitatively reflect the abundance of particular mRNA molecule in a
population.14
Transthoracic echocardiograph was performed with the HP Sonos
4500 (Hewlett-Packard Co, Palo Alto, CA) with a 10-MHz imaging
transducer, and the progress of cardiac hypertrophy was evaluated as
described previously.20
HSF1 Transgenic Mice
Construction of active form of human HSF1 transgene and generation of the hHSF1 transgenic mice have been previously described.15
Eight-week-old male transgenic and their wild-type littermates were
used. The transgenic mice were apparently healthy, and there were
no significant differences in the body weight (BW), heart weight to
BW ratio (HW/BW), or hemodynamic parameters, such as blood
pressure (BP) and heart rate (HR), between the transgenic mice and
wild-type littermates. Strong pressure overload was imposed on the
heart for 5 weeks by surgical constriction of transverse aorta (TAC)
as described previously.16
HSF1-Deficient Mice
The generation of HFS-1– deficient mice has been described previously.17 The HFS-1– deficient heterozygote mice were apparently
healthy. There were no significant differences in echocardiogram
parameters such as posterior wall thickness (PWTd) and fractional
shortening between the mutant mice and wild-type littermates. The
mice underwent 4 weeks of exercise (wheel running) or 1 week of
TAC as described above.
Histological Analysis
For histological analysis, hearts were fixed by perfusion with 10%
formalin. Fixed hearts were embedded in paraffin and sectioned at
4-␮m thickness. The myocyte cross-sectional diameter was measured in the sections stained with hematoxylin and eosin, and
suitable cross-sections were defined as having nearly circular capil-
Hemodynamic Measurements In Vivo
To measure hemodynamic effects of aortic constriction and exercise,
the right carotid artery was cannulated with a polyethylene catheter
(MILLAR, Houston, Tex). The transducer (model MP 5100, Baxter,
Deerfield, Ill) was connected to Mac Laboratory system (model
MacLab4/s, AD Instruments, Castle Hill, Australia), and BP, HR,
and LV end-diastolic pressure were measured.
Statistical Analysis
Data were shown as mean⫾SEM. Multiple group comparison was
performed by 1-way ANOVA, followed by the Bonferroni procedure
for comparison of means. Comparison between 2 groups were
analyzed by the 2-tailed Student’s t test or 2-way ANOVA. Values
of P⬍0.05 were considered statistically significant.
Results
Physiological Analysis
After 8 weeks of voluntary exercise or 5 weeks of pressure
overload, physiological analyses were performed in 13-weekold rats. Echocardiogram revealed that PWTd and LV enddiastolic dimension were larger in both groups compared with
those in the sham-operated group (Table I in the online data
supplement, available at http://circres.ahajournals.org). Sustained pressure overload developed cardiac hypertrophy more
profoundly than exercise (supplemental Table I). Fractional
shortening was decreased in the pressure-overload group but
not in the exercise group (supplemental Table I). In hemodynamic parameters, BP measured in the right carotid artery and
LV end-diastolic pressure were much higher in the pressureoverload group than the sham-operated group (supplemental
Table II). On the other hand, BP was significantly lower and
HR was less in the exercise model compared with the
pressure-overload model and the sham-operated model (sup-
Sakamoto et al
HSF1 in Cardiac Hypertrophy
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Figure 1. Histological analysis of rat
models. A through D, High-magnification
views of hematoxylin and eosin–stained
LV of sham-operated rats (A), pressureoverloaded rats (B), and exercised rats
(C). Scale bar⫽10 ␮m. D, Both pressure
overload (PO) and exercise (EX) induced
an increase in diameter of cardiomyocytes. *P⬍0.01 vs sham (n⫽8). E through
G, High-magnification views of Massonstained LV of sham-operated rats (E),
pressure-overloaded rats (F), and exercised rats (G). H, An increase in perivascular and interstitial fibrosis was
observed only in the pressure-overload
model. *P⬍0.01 vs sham (n⫽8).
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plemental Table II). In both cardiac hypertrophy models, LV
weight to BW ratio was increased (supplemental Table II).
The increase was more prominent in the pressure-overload
model than in the exercise model. Histological analysis
demonstrated that myocyte diameter was larger in both
hypertrophy models compared with the sham-operated model
(Figure 1A through 1D). In contrast, Masson staining revealed that cardiac fibrosis was significantly increased in
only the pressure-overload model but not in the exercise
model (Figure 1E through 1H). These results indicate that
chronic pressure overload but not exercise induces cardiac
fibrosis, thereby promoting maladaptive cardiac hypertrophy.
Expression Level of Hsp70 Protein and Activity of
HSF1 Were Elevated in
Exercise-Induced Hypertrophy
To determine the molecular difference between exercise- and
chronic pressure overload–induced hypertrophy, we performed DNA chip analysis. Expression levels of ⬇100 genes
were differently elevated in each model compared with the
sham-operated model. mRNA levels of fetal genes such as
atrial natriuretic peptide, brain natriuretic peptide, and skeletal ␣-actin were elevated ⬇2- to 3-fold in the pressureoverload model but not in the exercise model (Table 1). It is
important to note that expression level of the cardioprotective
TABLE 1.
DNA Chip Analysis
Sham
Pressure Overload
Exercise
ANP
807
2992
710
BNP
776
1552
656
Natriuretic factor
Organism defense
Hsp70
35
29
104
Hsp27
934
1315
2150
Muscle protein
Smooth muscle MHC
Skeletal ␣-actin
21
69
40
1985
4640
1393
Genes increased in rats with pathological (pressure overload) and physiological (exercise) cardiac hypertrophy compared with a sham-operated rat
(Sham). ANP indicates atrial natriuretic peptide; BNP, brain natriuretic peptide;
MHC, myosin heavy chain.
gene Hsp70 and Hsp27 was upregulated ⬇2- to 3-fold in the
exercise model but not in the pressure-overload model (Table 1).
We performed Northern blot analysis and Western blot
analysis to confirm the results of the DNA chip analysis.
Northern blot analysis revealed that mRNA levels of Hsp70
and Hsp27 were significantly increased in only the exercise
model (Figure 2A and data not shown). HSF1, a key molecule
regulating gene expression of Hsps, was also upregulated
only in the heart of the exercise model (Figure 2A). Protein
levels of inducible form of Hsp70 and HSF1 were significantly higher in the exercise model than those in the pressureoverload model and the sham-operated model (Figure 2B).
We next examined the activity of HSF1 by gel mobilityshift assay using a HSF1-binding DNA sequence. Shifted
band was more obvious in cardiac extracts prepared from
the exercise model than in those from the pressureoverload model and the sham-operated model (Figure 2C),
suggesting that HSF1 is activated in exercise-induced
hypertrophy.
Protective Role of HSF1 in Sustained Pressure
Overload–Induced Hypertrophy
To elucidate the role of HSF1 in cardiac hypertrophy, we
established the cardiac hypertrophy model by TAC in transgenic mice that express constitutively active HSF115 and
analyzed them 5 weeks after the operation. We also performed the same operation in their wild-type littermates as
controls. Expression of activated HSF1 was detected in the
heart of transgenic mice but not in wild-type mice (Figure
3A). Inducible Hsp70 expression was markedly increased in
the heart of transgenic mice compared with wild-type mice
(Figure 3A). Pressure studies revealed that there was no
difference in BP and HR between transgenic and wild-type
mice before the operation (data not shown). After the operation, BP was significantly increased in both transgenic mice
and wild-type mice to the same extent (Table 2). LV
end-diastolic pressure was significantly elevated in wild-type
mice, which was significantly ameliorated in transgenic mice
(Table 2). HW/BW was significantly increased in wild-type
mice, but the increases in HW/BW were significantly less in
transgenic mice than in wild-type mice (Table 2). Echocardiographic analysis demonstrated that an increase in PWTd
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December 8/22, 2006
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Figure 2. Expression of Hsp70 and HSF1
in the heart of rat models. A, Total RNA
was prepared from the heart of shamoperated rats, pressure-overloaded rats
(PO), or exercised rats (EX) and examined for Hsp70 and HSF1 expression by
Northern blot analysis. Ethidium bromide
staining of 18S was shown as a loading
control. Expression levels relative to
sham-operated rats were plotted in the
graph. *P⬍0.05, **P⬍0.01 vs sham;
†P⬍0.05, ††P⬍0.01 vs pressure overload (n⫽4). B, Whole-cell lysates were
prepared from the heart of shamoperated rats, pressure overloaded rats
(PO) or exercised rats (EX) and examined
for inducible Hsp70 and HSF1 expression by Western blot analysis. Expression levels relative to sham-operated rats
were plotted in the graph. *P⬍0.05 vs
sham; †P⬍0.05 vs PO (n⫽4). C, Nuclear
extracts from the hearts were subjected
to the gel mobility shift assay. Heat
shock response element was used as a
DNA probe. More prominent shifted
band was observed in the exercise
model than the pressure-overload and
sham-operated models. Same experiments were performed at least 3 times
and similar results were obtained.
by TAC was significantly attenuated in transgenic mice
compared with that in wild-type mice (Figure 3B). More
importantly, cardiac function was significantly better in
transgenic mice than in wild-type mice (Figure 3B), suggesting a protective role of HSF1 in cardiac hypertrophy.
Histological analysis showed that TAC induced marked
cardiomyocyte hypertrophy in wild-type mice after 5 weeks,
whereas transgenic mice developed less cardiomyocyte hypertrophy in response to sustained pressure overload than
wild-type mice (Figure 4A). Moreover, cardiac fibrosis was
significantly less in transgenic mice compared with wild-type
mice (Figure 4B). Because TAC has been reported to induce
death of cardiomyocytes,21 we examined death of cardiomyocytes in the heart by TUNEL. TUNEL-positive cells were
increased in the heart of wild-type mice compared with sham
mice (Figure 4C). The number of TUNEL-positive cells was
significantly less in the heart of transgenic mice compared
with that of wild-type mice (Figure 4C). Expression of
transforming growth factor-␤, a cytokine that is crucial for
tissue fibrosis,22 was markedly increased in the heart after
pressure overload (Figure 4D). This induction was significantly prevented by overexpression of HSF1. It has been
reported that HSF1 and Hsps suppress activation of transcription factors such as activator protein-1 and nuclear factor
␬B,23–25 both of which are known to upregulate proinflammatory cytokines including transforming growth factor-␤.
Thus, HSF1 may prevent cardiac fibrosis as well as cardiomyocyte death by antagonizing these proinflammatory pathways, thereby preventing the transition from adaptive to
maladaptive hypertrophy.
Figure 3. Sustained pressure overload–
induced hypertrophy in HSF1 transgenic
mice. A, Whole-cell lysates were prepared from the hearts of wild-type mice
(WT) and HSF1 transgenic mice (TG) and
examined for HSF1 and inducible Hsp70
expression by Western blot analysis.
Arrow indicates a constitutively active
form of HSF1. B, Pressure overload was
imposed on wild-type mice (WT-TAC)
and HSF1 transgenic mice (TG-TAC) by
5 weeks of TAC. Sham operation was
also imposed on wild-type mice (Sham).
Cardiac hypertrophy (PWTd) and systolic function (percentage of fractional shortening [%FS]) were examined 5 weeks after TAC by
echocardiogram. *P⬍0.05 vs sham, †P⬍0.05 vs WT-TAC (n⫽5).
Sakamoto et al
HSF1 in Cardiac Hypertrophy
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HSF1 Deficiency Impairs the Adaptive Response
to Exercise or Pressure Overload
hypertrophy, we produced the TAC model in HSF1⫹/⫺ mice
and their wild-type littermates and analyzed cardiac function
1 week later. In this adaptive phase, TAC increased expression of inducible Hsp70 in the heart of wild-type mice (Figure
6A). This induction was markedly attenuated in the heart of
HSF1⫹/⫺ mice (Figure 6A). Consistent with the results of our
rat model, expression of inducible Hsp70 was downregulated
in the maladaptive phase (supplemental Figure). Both
HSF1⫹/⫺ mice and wild-type mice developed cardiac hypertrophy to the degree similar to that of 4 weeks of exercise
(Figures 5C, 5D, 6B, and 6C). Cardiac function was preserved in wild-type mice, but it was significantly impaired in
HSF1⫹/⫺ mice (Figure 6C), further supporting a notion that
HSF1 has an important role in adaptive hypertrophy.
We next determined whether HSF1 has a protective role in
exercise-induced hypertrophy. We produced the exerciseinduced cardiac hypertrophy model in HSF1-deficient heterozygote (HSF1⫹/⫺) mice17 and compared them with their
wild-type littermates. Western blot analysis demonstrated that
expression of HSF1 was less in the heart of HSF1⫹/⫺ mice
than wild-type mice (Figure 5A). Expression of inducible
Hsp70 was increased in the heart of wild-type mice by
exercise, but this increase was attenuated in the heart of
HSF1⫹/⫺ mice (Figure 5B). There were no differences in
HW/BW and wall thickness (PWTd) between wild-type mice
and HSF1⫹/⫺ mice before exercise (Figure 5C and 5D). After
4 weeks of exercise, both mice developed cardiac hypertrophy to a similar extent (Figure 5C and 5D). In contrast to the
preserved function of wild-type mice, cardiac function of
HSF1⫹/⫺ mice was significantly reduced after exercise (Figure 5D), suggesting a critical role of HSF1 in the adaptive
mechanism of cardiac hypertrophy induced by exercise.
We noted that pressure overload by TAC induced adaptive
hypertrophy until 1 to 2 weeks after operation to preserve
cardiac function; however, this adaptive mechanism could not
protect the hypertrophied heart against sustained pressure
overload, resulting in systolic dysfunction at 4 to 5 weeks
(Figure 3B). To further test the role of HSF1 in adaptive
In the present study, we performed DNA chip analysis to
elucidate the molecular difference between exercise-induced
and sustained pressure overload–induced cardiac hypertrophy. We found that ⬇100 genes were differently elevated in
each hypertrophy model compared with the sham-operated
model. Among them, expression levels of Hsp70 and Hsp27
were elevated in only the heart of the exercise model, which
was confirmed by Northern and Western blot analysis. Hsps
such as Hsp70 and Hsp27 are ubiquitously expressed and its
expression is enhanced by various stresses such as heat
shock,26 reactive oxygen species,26 heavy metals,27 and inflammation.28 It has been reported that stimuli that result in
the transient induction of Hsps in the heart protect, to some
degree, against detrimental effects of ischemia/reperfusion
injury.29,30 Constitutive expression of Hsps in transgenic mice
efficiently recovers cardiac function after ischemia and reperfusion,31–33 whereas absence of Hsps leads to cardiac dysfunction and impairs stress response.34 Several studies have
reported that expression of Hsps is rapidly induced in the
heart in response to acute pressure overload, as well as
volume overload.35 However, there has been no report concerning expression of Hsps in the chronic stage of cardiac
TABLE 2.
Hemodynamic Parameters and Heart Weight
Sham
WT-TAC
102.3⫾7.3
153.0⫾5.6†
145.5⫾6.3†
622⫾32
602⫾15
588⫾18
LVEDP, mm Hg
7.8⫾1.8
16.6⫾2.2†
12.0⫾0.8†‡
HW, mg/BW (g)
4.72⫾0.16
6.74⫾0.29†
5.79⫾0.40*‡
BP, mm Hg
HR, bpm
TG-TAC
Sham indicates sham-operated mice; WT-TAC and TG-TAC, wild-type and
transgenic mice, respectively, receiving pressure overload by 5 weeks of TAC;
LVEDP, LV end-diastolic pressure. *P⬍0.05, †P⬍0.01 vs Sham; ‡P⬍0.05 vs
WT-TAC (n⫽7).
Discussion
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Figure 4. Histological analysis of the
heart after 5 weeks of TAC. A through C,
Pressure overload was imposed on wildtype mice (WT-TAC) and HSF1 transgenic mice (TG-TAC) by 5 weeks of TAC.
Sham operation was also imposed on
wild-type mice (Sham). The transverse
diameter of cardiomyocytes (A), the
extent of perivascular and interstitial
fibrosis (B), and the number of TUNELpositive cells/10 000 cardiomyocytes (C)
were estimated as described in Materials
and Methods. *P⬍0.05 vs sham,
†P⬍0.05 vs WT-TAC (n⫽5). D, Total
RNA was extracted from the hearts of
sham, WT-TAC, or TG-TAC and examined for expression of transforming
growth factor (TGF)-␤ by ribonuclease
protection assay. Expression levels relative to sham-operated mice were plotted
in the graph. *P⬍0.05 vs sham, †P⬍0.05
vs WT-TAC (n⫽4).
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December 8/22, 2006
Figure 5. Exercise-induced hypertrophy
in HSF1-deficient mice. A, Western blot
analysis for HSF1 in the heart of HSF1deficient heterozygote mice (KO) and
their wild-type littermates (WT). B, HSF1deficient heterozygote mice (KO-EX) and
their littermate wild-type mice (WT-EX)
were exercised for 4 weeks. Sham operation was also imposed on both types of
mice (KO-Sham and WT-Sham). Wholecell lysates were then prepared from the
hearts and examined for inducible Hsp70
expression by Western blot analysis. C,
HW/BW was measured before and after
exercise. *P⬍0.05 vs before exercise
(n⫽3– 4). D, Hypertrophy (PWTd) and
cardiac function (percentage of fractional
shortening [%FS]) were examined before
and after exercise by echocardiogram.
*P⬍0.01 vs before exercise, †P⬍0.01 vs
wild-type mice after exercise (n⫽4).
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hypertrophy. In this study, Hsp70 and Hsp27 were upregulated in the heart after exercise but not in the heart after
long-term pressure overload. These results suggest that exercise persistently upregulates expression of Hsps, thereby
inhibiting cardiac dysfunction associated with hypertrophy,
whereas pressure overload transiently induces expression of
Hsps and thus fails to prevent impaired function in the
chronic phase.
The transcription of Hsps genes is mainly controlled by
HSF1.9 –11 Impaired activation of HSF1 in the aged heart
results in diminished induction of Hsps by stressful stimuli
such as heat shock or ischemia. This impairment is associated
with reduced protective effects of mild heat shock or ischemia against subsequent severe ischemic stresses in the aged
heart.36 HSF1 deficiency has been reported to decrease
cardiac expression of Hsps,37 further supporting a critical role
of HSF1 in the regulation of Hsps expression. Although
pressure overload and ischemia/reperfusion injury have been
reported to rapidly induce HSF1 activation and Hsps expression in the heart,30,38 we found that HSF1 was activated in
only the heart of the exercise-induced hypertrophy model but
not in pressure overload–induced hypertrophy model at the
chronic stage, suggesting that exercise promotes sustained
activation of HSF1, which results in constitutive expression
of Hsps in the heart. It remains unclear how HSF1 is
downregulated under sustained pressure overload. Extracellular signal-regulated protein kinase (ERK) is known to
increase HSF1 activity.39 It is also reported that ERK is
activated by pressure overload and that its activation is
involved in the adaptive mechanism of hypertrophy.40 Moreover, we observed that ERK was activated in exerciseinduced hypertrophy but not in the chronic phase of pressure
overload–induced hypertrophy (M. Sakamoto and I. Komuro,
unpublished data, 2005), suggesting that the ERK signaling
pathway may participate in the activation of HSF1 in
exercise-induced hypertrophy and that its downregulation
may cause the maladaptive response to chronic pressure
overload by inactivating HSF1.
Constitutive activation of HSF1 prevented cardiac dysfunction as well as hypertrophy under chronic pressure
overload. Decreased activity of HSF1 did not exacerbate
hypertrophy but impaired systolic function in response to
exercise or acute pressure overload, a load that usually results
in adaptive hypertrophy. Thus, HSF1 activation may play an
essential role in inhibiting the transition from adaptive to
maladaptive hypertrophy rather than in regulating the size of
cardiomyocytes. The different manifestations of hypertrophy
between the exercise model and the long-term pressureoverload model might be attributable to the differences in the
continuity and degree of stimulus. In a recent report, Perrino
Figure 6. Acute pressure overload–induced hypertrophy in
HSF1-deficient mice. A, Pressure overload was imposed on
wild-type mice (WT-TAC) and HSF1-deficient mice (KO-TAC) by
1 week of TAC. Sham operation was also imposed on wild-type
mice (Sham). Whole-cell lysates were prepared from the hearts
of wild-type mice and HSF1-deficient mice and examined for
inducible Hsp70 expression by Western blot analysis. B,
HW/BW was measured 1 week after pressure overload. *P⬍0.01
vs sham (n⫽5–7). C, Hypertrophy (PWTd) and cardiac function
(percentage of fractional shortening [%FS]) were examined 1
week after pressure overload by echocardiogram. *P⬍0.01 vs
sham, †P⬍0.05 vs WT-TAC (n⫽4 to 7).
Sakamoto et al
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et al41 applied intermittent pressure overload to the hearts of
mice and tested the roles of duration and nature of the stress
on the development of cardiac failure. Despite a mild hypertrophic response, hearts exposed to intermittent pressure
overload displayed various pathological features including
diastolic dysfunction. Thus, the nature of the stress on the
heart, rather than its duration, is the key determinant of the
maladaptive phenotype. To test the role of the degree of
stimulus, we compared the effect of 1 week of TAC on
cardiac function with that of 4 weeks of exercise for the
following reasons. First, pressure overload by TAC induces
adaptive hypertrophy until 1 to 2 weeks after operation to
preserve cardiac function. Second, 4 weeks of exercise did
not induce cardiac dysfunction in wild-type mice. Third, 4
weeks of exercise induces cardiac hypertrophy to the degree
similar to that of 1 week of TAC. Finally, expression of
inducible Hsp 70 is upregulated in the heart of wild-type mice
in both conditions. We produced these 2 different models for
adaptive hypertrophy in HSF1⫹/⫺ mice and their wild-type
littermates. Cardiac function of HSF1⫹/⫺ mice was significantly impaired in both models compared with their wild-type
littermates. Moreover, expression of inducible Hsp 70 was
markedly reduced in the heart of HSF1⫹/⫺ mice. Although the
nature of 2 different stimuli may lead to the differential
molecular response, these results suggest a critical role of
HSF1 in adaptive hypertrophy. Downregulation of HSF1
activity in the chronic phase may be a key factor to promote
the transition from adaptive to maladaptive hypertrophy.
Alternatively, the different manifestations of hypertrophy
(adaptive or maladaptive) may be partly attributed to the
difference in HSF1 activity between the exercise model and
the chronic pressure-overload model because reducing its
activity caused maladaptive hypertrophy in both conditions.
Sustained pressure overload induced cardiomyocyte death
and fibrosis in the heart, both of which have been reported to
cause heart failure.42 In the heart of HSF1 transgenic mice,
the number of TUNEL-positive cardiomyocytes and the
extent of fibrosis were significantly less than those of
wild-type mice. These protective effects may be attributable
to the functions of Hsps in protein folding and degradation. In
addition to such well-known functions, accumulating evidence indicates that different Hsps directly act on the cell
death machinery and inhibit the signaling pathway for cell
death at various points.43 For example, Hsp27 binds to
cytochrome c and prevent it binding to Apaf-1,44 whereas
Hsp70 prevents Apaf-1 from recruiting pro– caspase-9,45
thereby inhibiting apoptotic cell death. Consequently, induction of HSF1 activity rather than individual Hsps may be
more effective to protect the heart from severe stresses and
will be an attractive therapeutic target in cardiovascular
pathophysiology.
Sources of Funding
This work was supported by a grant-in-aid for scientific research
from the Ministry of Education, Science, Sports, and Culture; health
and labor sciences research grants from the Ministry of Health,
Labor and Welfare; and a grant for research on life science from
Uehara Memorial Foundation, Japan.
HSF1 in Cardiac Hypertrophy
1417
Disclosures
None.
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Upregulation of Heat Shock Transcription Factor 1 Plays a Critical Role in Adaptive
Cardiac Hypertrophy
Masaya Sakamoto, Tohru Minamino, Haruhiro Toko, Yosuke Kayama, Yunzeng Zou, Masanori
Sano, Eiichi Takaki, Teruhiko Aoyagi, Katsuyoshi Tojo, Naoko Tajima, Akira Nakai, Hiroyuki
Aburatani and Issei Komuro
Circ Res. 2006;99:1411-1418; originally published online November 9, 2006;
doi: 10.1161/01.RES.0000252345.80198.97
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2006 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
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Online Supplement
Sakamoto et al.
HSF1 in cardiac hypertrophy
Supplemental Table 1
Echocardiographic analysis
Sham
PO
EX
PWTd (mm)
1.54±0.06
2.19±0.13**†
1.76±0.02*
LVDd (mm)
6.6±0.1
7.61±0.16*
7.14±0.02*
FS (%)
49.43±1.10
41.20±2.33**†
53.42±0.94
Sham, sham-operated rat; PO, pressure-overloaded rat; EX, exercised rat; PWTd,
posterior wall thickness at diastole; LVDd, left ventricular dimension at diastole; FS,
fractional shortening. *p<0.05, **p<0.01 vs. Sham; †p<0.01 vs. EX (n=8).
Online Supplement
Sakamoto et al.
HSF1 in cardiac hypertrophy
Supplemental Table 2
Hemodynamic parameters and heart weight
Sham
PO
EX
BP (mmHg)
99.3±7.2
144.6±9.0**†
84.4±4.3*
HR (bpm)
318±19
323±15†
250±11**
LVEDP (mmHg)
0.85±0.05
4.77±0.18*
1.12±0.10
LVW (mg)/BW(g)
1.83±0.05
2.77±0.19 **†
2.17±0.10*
Sham, sham-operated rat; PO, pressure-overloaded rats; EX, exercised rats; BP, blood
pressure; HR, heart rate; LVEDP, left ventricular end-diastolic pressure; LVW, left
ventricular weight; BW, body weight. *p<0.05, **p<0.01 vs. Sham; †p<0.05 vs. EX
(n=8).
Online Supplement
Sakamoto et al.
HSF1 in cardiac hypertrophy
Supplemental Figure
Expression of inducible Hsp70 after pressure overload.
Pressure overload was imposed on wild-type mice. Whole cell lysates were extracted
from the hearts at the indicated time points and examined for expression of inducible
Hsp70 by Western blot analysis.
Supplementary
figure
Supplemental
Figure
TAC
Hsp70
Actin
0
3
7
14
28
(days)