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Journal of Molecular and Cellular Cardiology 43 (2007) 616 – 626
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Original article

Hyperthermia treatment prevents angiotensin II-mediated atrial fibrosis
and fibrillation via induction of heat-shock protein 72
Osamu Wakisaka a , Naohiko Takahashi a,⁎, Tetsuji Shinohara a , Tatsuhiko Ooie b ,Mikiko Nakagawa b , Hidetoshi Yonemochi b , Masahide Hara a , Tatsuo Shimada c ,Tetsunori Saikawa b , Hironobu Yoshimatsu aaDepartment of Internal Medicine 1, Faculty of Medicine, Oita University, Oita, 1-1 Idaigaoka, Hasama, Oita 879-5593, Japanb
Department of Cardiovascular Science, Faculty of Medicine, Oita University, Oita, Japanc
Department of Health Science, School of Nursing, Oita University, Oita, Japan
Received 25 June 2007; received in revised form 19 July 2007; accepted 2 August 2007
Available online 16 August 2007

Abstract
We tested the hypothesis that atrial fibrosis and atrial fibrillation (AF) evoked by angiotensin II (AII) could be prevented by the induction ofheat-shock protein 72 (HSP72) by hyperthermia (HT). In cultured atrial fibroblasts isolated from male Sprague–Dawley rats, HT (42 °C) wasapplied for 30 min. AII (100 nmol/L) was added to the medium 8 h later. HT induced the expression of HSP72, which was associated with theattenuation of AII-induced extracellular signal-regulated kinase (ERK1/ERK2) phosphorylation, α-smooth muscle actin (α-SMA) expression,transforming growth factor-β1 secretion, collagen synthesis, and expression of collagen type I and tissue inhibitor of metalloproteinases-1. A smallinterfering RNA targeting HSP72 abolished these anti-fibrotic effects of HT. In male Sprague–Dawley rats in vivo, an osmotic mini-pump was
subcutaneously implanted for continuous infusion of AII (400 ng/kg/min). Whole-body HT (43 °C, 20 min) was applied 24 h before and 7, 14,
and 21 days after the start of the AII infusion. Repeated HT led to the induction of HSP72 expression, which resulted in an attenuation of AIIinduced left atrial fibrosis. In an electrophysiological study using isolated perfused heart, continuous AII caused slowing of interatrial conduction
without affecting atrial refractoriness. In AII-treated hearts, extrastimuli from the right atrial appendage resulted in a high incidence of repetitive
atrial responses, which were suppressed by treatment with HT. Our results suggest that HT treatment is effective in suppressing AII-mediated atrial
fibrosis and AF via induction of HSP72 at least in parts, and is thus expected to be a novel strategy for prevention of AF.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Atrial fibrillation; Fibrosis; Heat-shock response

1. Introduction
Atrial fibrillation (AF) is the most common arrhythmia in theclinical setting and is promoted by activation of the renin–
angiotensin system [1,2]. Angiotensin II (AII) has been reported
to participate in the maintenance of AF by the induction of atrial
structural remodeling, which is characterized by interstitial
fibrosis [3,4]. Two-thirds of the cell population of the heart iscomposed of fibroblasts [5]. In response to the infusion of AII, it
has been reported that fibroblasts proliferate, and myofibroblasts⁎ Corresponding author. Tel.: +81 97 586 5793; fax: +81 97 549 4480.
E-mail address: takanao@med.oita-u.ac.jp (N. Takahashi).
0022-2828/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.yjmcc.2007.08.005

that contain α-smooth muscle actin (α-SMA), a contractile
protein, appear, in association with an increase in collagenvolume and interstitial fibrosis [6]. The phosphorylation of
extracellular signal-regulated kinases (ERK1/ERK2) has beenshown to play a critical role in these processes [1].
Heat-shock protein 72 (HSP72) is an important endogenous
protective protein that functions as a molecular chaperone [7],and we have shown that induction of cardiac HSP72 by hyperthermia (HT) or geranylgeranylacetone (GGA) protects the rat
heart against ischemia/reperfusion injury [8–11]. Interesting
clinical observations of the suppressive effects of HSP72 on AF
have been recently reported. Mandal et al. [12] demonstrated, inpatients undergoing elective coronary artery bypass surgery,O. Wakisaka et al. / Journal of Molecular and Cellular Cardiology 43 (2007) 616–626

that the preoperative HSP72 content in right atrial tissue
obtained at surgery was higher in patients who did not develop
postoperative AF than in those who developed AF. Inductionsof heat-shock responses, particularly HSP27 induction, havebeen shown experimentally to protect the heart against atrial
fibrillation, although the specific roles of HSP72 have not yet
been elucidated [13,14].
Based on these observations, we tested the hypotheses: (1)that HT treatment could suppress the intracellular signals that
stimulate fibrosis in cultured rat atrial fibroblasts exposed to
AII via induction of HSP72; and (2) that HT treatment
induction could prevent atrial fibrosis and reduce atrial vulnerability to developing AF in rats exposed in vivo to acontinuous subcutaneous infusion of AII.

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cells were equilibrated in serum-free DMEM 24 h before
incubation under the conditions of HT (42 °C for 30 min) or
normothermia (NT, 37 °C for 30 min). Preliminary observations
had demonstrated that HSP72 expression reached a peak 8 h after
HT. Therefore, 8 h after each thermal treatment, some fibroblasts
were collected to assess HSP72 expression. AII (100 nmol/L) or
vehicle was added to the medium of the remaining cells.
Incubated cells were collected 8 min after AII addition for theevaluation of ERK1/ERK2 phosphorylation, and 24 h after AII
addition for the evaluation of the expression of α-SMA, collagentype I, metalloproteinase-1 (MMP-1), and tissue inhibitor of
metalloproteinases-1 (TIMP-1) by Western blot analysis. The
expression of endoplasmic reticulum (ER) chaperone including
glucose regulated proteins 78 and 94 (GRP78 and GRP94) was
also evaluated by Western blot analysis.

2. Methods
2.2. Western immunoblotting
All experimental procedures were in accordance with theguidelines of the Physiological Society of Oita University,
Japan, for the care and use of laboratory animals, which follow
the guidelines of the National Institute of Health (USA).
2.1. Isolation and culture of adult rat atrial cardiac fibroblasts,
small interfering RNA targeting HSP72, and thermal treatment
Adult male Sprague–Dawley rats (250 to 300 g) wereanesthetized with sodium pentobarbital (50 mg/kg, IP) and theleft atrium (LA) was removed and minced in phosphatebuffered saline (PBS) [15,16]. The pieces were washed threetimes with PBS, incubated in 0.02% EDTA for 5 min at 37 °C
with shaking, and centrifuged at 500 rpm for 2 min to remove
EDTA. The pieces were then incubated in 5 mL of Hanks’balanced salt solution (HBSS) containing collagenase at aconcentration of 2 mg/mL (Type IV; Cooper Biochemical) for
10 min in a 37 °C water bath shaken at 120 rpm. The
supernatant was discarded, and the remaining pieces of tissue
were treated with dispase (1000 IU/mL; Godo Shusei) in 5 mLof Ca2+- and Mg2+-free HBSS, with stirring for 20 min at 37 °C.
Dispase treatment was repeated twice more, after which thefree-floating cells were stored in Dulbecco's modified Eagle's
medium (DMEM). The cardiac myocytes were pelleted bycentrifugation at 200 rpm for 2 min, and the supernatantcontaining the fibroblasts was collected. The fibroblasts werepelleted at 1000 rpm for 10 min and resuspended in DMEM
supplemented with kanamycin and 10% fetal bovine serum(FBS). After a 30-min period of attachment to of the fibroblasts
to tissue culture plates, cells that were weakly attached or
unattached were rinsed off and discarded. The fibroblasts were
cultured in DMEM containing 10% FBS at 37 °C under 5%
CO2 and 95% room air. After 2 to 3 days, confluent cultures
were passaged by trypsinization and replated at a split ratio of
1:3. Fibroblasts at passage 2 were used in the experiments.
Some of the atrial fibroblasts were transfected with smallinterfering RNA (siRNA) targeting HSP72 (HSP72 siRNA) at aconcentration of 100 nmol/L with siPORT NeoFX Transfection(Ambion) [17]. Control cells were transfected with negative
control siRNA. Twenty-four hours after siRNA transfection, theWhole cell lysates were prepared in cell lysis buffer andhomogenized by sonication [8–10]. Equal amounts of protein,assayed using the Bradford method [18], were loaded on 10%
SDS/PAGE and transferred electrophoretically to a polyvinylidene difluoride membrane. After transfer and blocking with
5% nonfat milk, the membranes were incubated with antiHSP72 monoclonal antibody (Stressgen), anti-Phospho-p44/42
MAP kinase (Thr202/Tyr204) (phospho-ERK1/ERK2) antibody (Cell Signaling), anti-p44/42 MAP kinase (ERK1/ERK2)
antibody, monoclonal anti-α-SMA antibody (Sigma), anticollagen type I antibody (Santa Cruz), anti-MMP-1 antibody(Oncogene), anti-TIMP-1 antibody (Santa Cruz), anti-GRP78
and GRP94 antibody (Stressgen), or monoclonal anti-vimentin
antibody (Sigma) overnight at 4 °C. The proteins were detectedby enhanced chemiluminescence with exposure to Hyperfilm(Amersham Pharmacia). The amount of protein on the
immunoblots was quantified using National Institutes of Healthsoftware (NIH Image).
2.3. Inositol phosphate production
Atrial fibroblasts were labeled for 18 h with myo-[3H]
inositol (2 μCi/mL; GE Healthcare) before HT or NT treatment.
Eight hours after each thermal treatment, the cells were washedin medium containing 10 mmol/L LiCl for 20 min, and thenincubated with AII (10 or 100 nmol/L) or vehicle for 30 min.
Some labeled cells were incubated with candesartan, an AII type
1 (AT1) receptor antagonist, at a concentration of 10 nmol/L for
5 h, followed by the addition of AII (100 nmol/L). Inositol
phosphates (IP) and total inositol fractions were resolved on a
Dowex AG1-X8 formate column (Bio-Rad), and IP accumulation was estimated by determining the ratio of IP radioactivity to
the sum of IP plus inositol radioactivity [19].
2.4. Immunocytochemistry
Atrial fibroblasts cultured on glass coverslips (Lab-Tek II,
Nalge Nunc Int) were transfected with siRNA for 24 h, incubatedwith serum-free DMEM for 24 h, and then incubated under the618

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conditions of HT or NT. AII (100 nmol/L) or vehicle wassubsequently added to the medium. Twenty-four hours after the
addition of AII or vehicle, the atrial fibroblasts were fixed in 4%
paraformaldehyde for 30 min and washed twice with PBS. The
cells were permeabilized in 0.5% Triton X-100/PBS for 30 min,blocked with 1% goat serum/PBS for 1 h, and incubated with
anti-α-SMA antibody (Sigma) overnight. After washing twicewith PBS, the atrial fibroblasts were incubated with anti-mouse
IgG conjugated to FITC (fluorescein isothiocyanate, Sigma) andmounted in Vectashield mounting media containing DAPI (4′,6diamino-2-phenylindole) for nuclear staining (Vector Laboratories, Burlingame) [16].

zine (1.5 mg/kg/day), hydrochrolothiazide (5 mg/kg/day) andreserpine (0.1 mg/kg/day) was mixed into the drinking water ofa subgroup of AII-treated rats given NT (AII-NT-HHR group)[22]. LA was removed from the rats at days 1, 8, 15, 22, and 28,
and frozen in liquid nitrogen and kept at − 80 °C. They wereused to assay the expression of HSP72 by Western blotting or
measurement of atrial natriuretic peptide (ANP) mRNA by realtime quantitative RT-PCR. At day 28, hemodynamic parameters
were measured. Briefly, rats were anesthetized with sodium
pentobarbital (50 mg/kg, IP). A PE-50 tube was placed via theleft ventricular (LV) apex into the LV cavity. Hemodynamic
parameters were analyzed utilizing Chart v5.3 software (ADInstruments).

2.5. TGF-β1 secretion
2.8. Real-time quantitative RT-PCR
The secretion of transforming growth factor β1 (TGF-β1)
from atrial fibroblasts 24 h after AII addition was measured in
the supernatant using a TGF-β1 quantitative ELISA kit (R&D
systems Inc.) [20].

Atrial fibroblasts were transfected in the presence or absenceof HSP72 siRNA for 24 h and incubated with serum-free
DMEM for 18 h in medium containing L-[3H]proline (1 μCi/mL;
PerkinElmer Life and Analytical Science), followed by HT or
NT treatment. Eight hours later, AII (100 nmol/L) or vehicle wasadded to the medium for 24 h, and cells were collected. The
supernatant was replaced with ice-cold 10% trichloroacetic acidfor 20 min at 4 °C. The precipitate was rinsed with deionizedwater, and then solubilized in 0.3 N NaOH-0.1% SDS for 2 h at
37 °C. The cell lysate was added to liquid scintillant, and theincorporated radioactivity was measured [21].

ANP mRNAwas amplified by PCR and quantified using realtime quantitative PCR [23]. In brief, total cellular RNA wasprepared from LA using TRIzol (Lifetech). The cDNA wassynthesized from 150 ng of total RNA using a ReverTra-Dashreverse transcriptase kit (Toyobo). The sequences of the specificprimers were as follows: ANP (sense), 5′-ATGGGCTCCTTCTCCATCACC-3′; ANP (anti-sense), 5′-TCCGCTCTGGGCTCCAATCCTGT-3 ; ribosomal 28S (sense), GAATCCGCTAAGGAGTGTGTAACA; ribosomal 28S (anti-sense), CTCCAGCGCCATCCATTT. A primer for ribosomal RNA was used
as an internal control. Using an ABI PRISM 7000 sequence
detector (Applied Biosystems), PCR amplification was performed in a 50 μL volume containing 100 ng cDNA template in
PCR master mix (Roche). ANP mRNA and ribosomal RNA
values were calculated from standard curves obtained using twofold serial dilutions of cDNA from the tissues, and ANP mRNAamounts were normalized to ribosomal RNA.

2.7. Continuous infusion of AII and thermal treatment

2.9. Electrophysiological studies

An osmotic mini-pump (Alzet 2ML4, Alzet) was implanted
subcutaneously into male Sprague–Dawley rats weighing 250
to 300 g anesthetized with sodium pentobarbital (50 mg/kg, IP),for constant infusion of AII or vehicle [6]. Twenty-four hours
before the start of AII or vehicle infusion (day 0), HT (42 °C for
20 min) or NT (37 °C for 20 min) was applied as previously
described [8]. Briefly, rats were anesthetized with pentobarbital(20 mg/kg, IP) and placed, with their heads on a pillow to avoid
aspiration of water, for 20 min in a bath in which the watertemperature was maintained at 43 °C. Rectal temperature wasmonitored throughout the thermal treatment experiments.
Application of HT at 43 °C elevated the rectal temperature to
41 °C within 10 min and the temperature remained between 41
and 42 °C during HT application. In AII-treated rats, AII was
infused at a constant rate of 400 ng/kg/min. The thermal
treatment was repeated on days 7, 14, and 21. Systolic bloodpressure (SBP) was measured by tail cuff on days 1, 8, 15, 22,
and 28. Our preliminary experiments had revealed that AIItreated rats given repeated HT (AII-HT group) showed areduction in SBP compared with AII-treated rats given repeated
NT (AII-NT group). Therefore, a cocktail containing hydrala-

On day 28, hearts were isolated and perfused retrogradely
using a Langendorff apparatus with Krebs–Henseleit buffer (inmmol/L: NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4
1.2, NaHCO3 25.0, glucose 11.9; pH 7.4) equilibrated with a
95% O2/5% CO2 gas mixture at 36.5 °C and at a constantpressure of 75 mm Hg. Two sets of Teflon-coated (except theirtips) silver bipolar electrodes, each with an interelectrodedistance of 1 mm, were placed on the appendages of the rightatrium (RA) and LA. The distance between the electrodes on the
RA and LA was set at 10 mm. The effective refractory periods(ERP) of the RA and LA were measured by the S2 extrastimulus
method using 8 regularly paced beats with cycle lengths (CL) of
200, 150, 120, and 90 ms and four times the threshold current
for 2 ms. The interatrial conduction time (IACT) was measured
during RA pacing at CL of 200, 150, 120, and 90 ms. AF
vulnerability was tested by the S3 extrastimulus method. Theintervals of S1 to S2 and S2 to S3 were the same anddecremented from 100 ms to the ERP of the RA. Following
extrastimuli, we evaluated the appearance of a repetitive atrial
response (RAR), defined as rapid, irregular atrial excitations
continuing over six beats.

2.6. Collagen synthesis

O. Wakisaka et al. / Journal of Molecular and Cellular Cardiology 43 (2007) 616–626

2.10. Histological studies
LA was carefully removed, fixed in 10% phosphate-bufferedformalin, and embedded in paraffin. Deparaffined sections(5 μm) were stained with Masson trichrome. Microscopic
images were digitized using Photoshop 7.0 (Adobe) andanalyzed using NIH Image. Connective tissue was differentiated on the basis of its color and expressed as a percentage ofthe reference tissue area.

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of IP plus inositol radioactivity, the hemodynamic parameters,
ANP mRNA expression, and the percentages of fibrosis, ERP,
and IACT were analyzed using one-way ANOVA followed by
the Bonferroni–Dunn test. The incidence of RAR was analyzed
by the Chi-square test. A P value of b0.05 was considered
statistically significant.
3. Results
3.1. HSP72 expression in atrial fibroblasts

2.11. Statistical analysis
Data are expressed as the mean ± SE. Serial changes in
SBP were analyzed by two-way ANOVA followed by the
Bonferroni–Donn test, unless otherwise specified. The relative
density of each protein, the relative incorporated radioactivityof [3H]proline, the relative ratio of IP radioactivity to the sum

Fig. 1A shows representative bands of HSP72 protein in the
fibroblasts and the relative density, assessed 8 h after eachthermal treatment. When compared with NT-treated cells, HT
resulted in a 6.1-fold increase in HSP72 content (P b 0.01, data
not shown). Pretreatment with HSP72 siRNA reduced HTinduced HSP72 protein levels by approximately 60% (P b 0.01).

Fig. 1. (A) Expression of heat-shock protein 72 (HSP72) in atrial fibroblasts analyzed by Western blot. Above: Representative bands of HSP72 8 h after the applicationof hyperthermia (HT, left) and HT in the presence of HSP72 siRNA. Vimentin is an internal standard. Below: Quantitative expression of HSP72 (relative density). (B)Angiotensin II (AII)-induced inositol phosphate (IP) production in atrial fibroblasts. Data are relative radioactivity. (C) Extracellular signal-regulated kinase 1/2(ERK1/ERK2) phosphorylation in atrial fibroblasts analyzed by Western blot. Above: Representative bands of phosphorylated-ERK 1/2 (P-ERK 1/2, top) and total
ERK 1/2 (bottom). Below: Quantitative expression of P-ERK2 (relative density). Data are mean ± SE. In each experiment (A–C), four independent cultures were
evaluated. ∗P b 0.05, ∗∗P b 0.01, NS = not significant.

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O. Wakisaka et al. / Journal of Molecular and Cellular Cardiology 43 (2007) 616–626

3.2. Activation of AT1 receptor
The application of AII (10 or 100 nmol/L for 30 min)increased IP production in the fibroblasts (P b 0.01). While
pretreatment with HT did not inhibit AII-induced increases in IP
production, pretreatment with candesartan (10 nmol/L) suppressed AII-induced increases in IP production (Fig. 1B).

treated cells, AII induced a 1.5-fold increase in α-SMA content(P b 0.01). HT attenuated the AII-induced α-SMA expression(P b 0.05). Pretreatment with HSP72 siRNA abolished the
suppressive effects of HT on AII-induced α-SMA expression(P b 0.05).
3.5. TGF-β1 secretion, collagen synthesis, and expression ofcollagen type I, MMP-1, TIMP-1, and ER chaperone

3.3. ERK1/ERK2 phosphorylation
Fig. 1C demonstrates the phosphorylation of ERK1/ERK2
and the relative density of phosphorylated ERK2 in the
fibroblasts, when assessed 8 min after the addition of AII to
the culture medium. In NT-treated cells, AII enhanced thephosphorylation of ERK1 and ERK2 2.3-fold (P b 0.01) and
2.5-fold (P b 0.01), respectively. The increases in ERK1/
ERK2 phosphorylation were attenuated in HT-treated cells(P b 0.05). Pretreatment with HSP72 siRNA abolished the
suppressive effects of HT on AII-induced ERK1/ERK 2
phosphorylation (P b 0.05, relative densities for ERK1 are notshown).
3.4. α-SMA expression
Fig. 2A illustrates α-SMA expression in fibroblasts byimmunocytochemical analysis using laser microscopy. In NTtreated cells, AII caused a remarkable increase in expression of
α-SMA in the cytoplasm, when assessed 24 h after AII
application. Pretreatment with HT attenuated AII-induced αSMA expression; the effects were abolished by pretreatment
with HSP72 siRNA. Fig. 2B shows representative bands and therelative density of α-SMA by Western blot analysis. In NT-

When assessed 24 h after the addition of AII, TGF-β1
secretion had increased by 45% (P b 0.01, Fig. 3A). The
increase in AII-induced TGF-β1 secretion was attenuated by
HT (P b 0.05). Pretreatment with HSP72 siRNA abolished the
suppressive effects of HT on TGF-β1 secretion (P b 0.05). Fig.
3B shows collagen synthesis evaluated by proline incorporation. In NT-treated cells, AII increased collagen synthesis by
1.4-fold (P b 0.01). HT attenuated the AII-induced increase incollagen synthesis (P b 0.01); the effect was counteracted by
pretreatment with HSP72 siRNA (P b 0.05). Figs. 3C–E show
representative expression and the relative densities ofcollagen type I, MMP-1, and TIMP-1, respectively. In NTtreated cells, addition of AII resulted in a 2.4-fold increasein collagen type I content (P b 0.01). Pretreatment with HT
attenuated the AII-induced collagen type I expression(P b 0.01); this effect was counteracted by pretreatment with
HSP72 siRNA (P b 0.05). As shown in Fig. 3D, the expression of MMP-1 was not significantly influenced by AII,HT, or HSP72 siRNA. In contrast, an AII-induced increase in
TIMP-1 expression (P b 0.01) was attenuated by pretreatment
with HT (P b 0.05, Fig. 3E). The expression of ER chaperone,
represented by GRP78 and GRP94, was also evaluated (Figs.
3F and G). Both GRP78 and GRP94 were upregulated by

Fig. 2. Expression of α-smooth muscle actin (α-SMA) in atrial cardiac fibroblasts. (A) Immunocytochemical staining of atrial fibroblasts (passage 2) using specificα-SMA primary antibodies. (B) Above: Representative bands of α-SMA by Western blot analysis. Vimentin is an internal standard. Below: Quantitative expression of
α-SMA protein (relative density). AII = angiotensin II. HT = hyperthermia. Data are mean ± SE. Four independent cultures were evaluated. ∗P b 0.05, ∗∗P b 0.01,
NS = not significant.

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Fig. 3. (A) Transforming growth factor-β1 (TGF-β1) secretion. (B) [3H] proline incorporation. (C–G) Representative bands and quantitative expressions (relative
density) of collagen type I, metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinases-1 (TIMP-1), glucose regulated proteins 78 (GRP78), and GRP94.
AII = angiotensin II. HT = hyperthermia. Data are mean ± SE. In each experiment (A–G), four independent cultures were evaluated. ∗P b 0.05, ∗∗P b 0.01, NS = notsignificant.

AII, while the pretreatment with HT did not influence theexpression levels.
3.6. Time courses of SBP and cardiac HSP72 expression, andhemodynamic parameters on day 28

3.8. Atrial fibrosis
Fig. 5 shows representative histological sections of the LA
isolated from the three groups. Compared with the control group(Fig. 5A), extensive and heterogeneous interstitial fibrosis,

Fig. 4 shows the time courses of SBP and cardiac HSP72
expression in the LA (days 1, 8, 15, 22, and 28). Repeated HT
application (AII-HT group) effectively induced HSP72 expression in the LA (days 1, 8, 15, and 22), when compared with the
AII-NT-HHR group (P b 0.05, by ANOVA). Hemodynamic
parameters on day 28 are show in Table 1. Body weight andventricular weight were lower, but the ratio of ventricular/bodyweight was greater, in the AII-NT-HHR group compared with
the NT-treated control group (P b 0.05). With respect to thehemodynamic parameters, LV pressure, dP/dt max, dP/dt min,LV end diastolic pressure (LVEDP), and LV developed pressure(LVDP) were greater in the AII-NT-HHR group compared with
the NT-treated control group (P b 0.01). HT did not affect theweight or the hemodynamic parameters.
3.7. ANP mRNA expression
In the AII-NT-HHR group, ANP mRNA expression in LA
was increased to 177% when compared with the control group(100%, P b 0.01); this effect was suppressed by HT (133%,
P b 0.05, Table 1).

Fig. 4. Time course of systolic blood pressure (SBP) and HSP72 expression.
Whole body hyperthermia (HT) or normothermia (NT) was applied at days 0, 7,
14, and 21. SBP was measured at days 0, 7, 14, 21, and 28 after the start ofcontinuous AII infusion (400 ng/kg/min). In the panel, representative bands of
HSP72 protein in the AII-NT-HHR group and the AII-HT group at days 1, 8, 15,
22, and 28 are shown. Data points represent means ± SE. n = 7–8 in each group.

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O. Wakisaka et al. / Journal of Molecular and Cellular Cardiology 43 (2007) 616–626

Table 1
Characteristic and hemodynamic parameters, and tissue ANP mRNA expression
in the control, AII-NT-HHR, and AII-HT groups

Body weight (BW, g)
Ventricular weight (VW, g)
VW(mg)/BW(g)
LV pressure (mm Hg)
dP/dt max (mm Hg/s)
dP/dt min (mm Hg/s)
LV end diastolic pressure(mm Hg)
LV developed pressure(mm Hg)Left atrial ANP mRNA(% control)

Control(n = 6)

AII-NT-HHR(n = 6)

AII-HT(n = 6)452.5 ± 11.3
1.10 ± 0.02
2.45 ± 0.08
108.5 ± 3.6
4399 ± 216− 3650 ± 239
6.7 ± 0.6

296.4 ± 6.9*
0.98 ± 0.01*
3.33 ± 0.07*
213.9 ± 3.8**8709 ± 229**− 6024 ± 79**7.2 ± 0.3**

300 ± 5.6*
0.98 ± 0.02*
3.25 ± 0.07*
211.5 ± 3.1**
8789 ± 536**− 5922 ± 341**
7.2 ± 0.2**

101.7 ± 3.6

206.5 ± 4.0**

204.2 ± 3.3**

100 ± 10177.5 ± 18.9**133.0 ± 10.6#

Data are mean ± SEM. *P b 0.05, **P b 0.01 vs. control. #P b 0.05 vs. AII-NTHHR.
AII = angiotensin II; HHR = cocktail constituted with hydralazine (1.5 mg/kg/day), hydrochrolothiazide (5 mg/kg/day), and reserpine (0.1 mg/kg/day);
HT = hyperthermia; LV = left ventricular.

demonstrated using Masson trichrome staining, was observed in
the AII-NT-HHR group (P b 0.01, Fig. 5B). The interstitial
fibrosis induced by AII was attenuated in the AII-HT group(P b 0.05, Figs. 5C and D).

3.9. Electrophysiological studies
Figs. 6A and B depict the ERP of the RA and LA determinedat the four basic CL (BCL) of 200, 150, 120 and 90 ms,
respectively. In both the RA and the LA, the ERP in the control,
AII-NT-HHR, and AII-HT groups were slightly shortened witha reduction in BCL. However, no significant differences in ERP
were observed among the three experimental groups. The IACT
was also evaluated at the same four BCL (Fig. 6C). Comparedwith the control group, the IACT in the AII-NT-HHR group was
prolonged at all BCL tested (P b 0.05 for each CL). This
prolongation was not observed in the AII-HT group. Fig. 7Ashows two representative RAR induced by the S3 extrastimuli
that were observed in the AII-NT-HHR group, while Fig. 7Bshows the incidence of RAR in the three groups. RARs were
never induced in hearts from the control group. In the AII-NTHHR group, the S3 extrastimuli induced RAR in seven out ofeight rats (88%). In the AII-HT group, the induction of RAR
was observed in three out of eight rats (38%). Thus, pretreatment with HT resulted in a reduction in the incidence of
RAR in the AII-treated hearts (P b 0.05).
4. Discussion
In the present study, using cultured fibroblasts isolated fromthe LA of adult male rats, we observed that HT induced theFig. 5. Histology of the left atrial (LA) free wall. (A–C) Representative atrial tissue sections stained by Masson trichrome staining. (D) Histogram of mean interstitial
fibrosis of the LA in the control, AII-HT and AII-NT-HHR groups. Data are mean ± SE. n = 7–8 in each group.

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Fig. 6. Electrophysiological characteristics. (A) Effective refractory period (ERP) of the right atrium (RA). (B) ERP of the left atrium (LA). (C) Interatrial conduction
time (IACT). ERP and IACT were measured at the basic cycle lengths (BCLs) of 200, 150, 120, and 90 ms. Data are mean ± SE. n = 7–8 in each group. ∗P b 0.05 vs.
control and AII-HT groups.

expression of HSP72, which was associated with the attenuationof AII-induced fibrotic signals, including ERK1/ERK2 phosphorylation. The siRNA targeting HSP72 abolished the most of
the antifibrotic effects produced by HT, i.e. ERK1/ERK2
phosphorylation, α-SMA expression, TGF-β1 secretion, collagen synthesis, and collagen type 1 expression, suggesting asignificant role of HSP72 in antagonizing against AII-induced
promotion of fibrotic processes. In rats in vivo, repeated wholebody HT induced atrial HSP72 expression and resulted in theattenuation of AII-induced LA fibrosis. In isolated-perfusedhearts, continuous AII infusion caused interatrial conduction
slowing without affecting atrial ERP. We evaluated the atrial

vulnerability by the incidence of RARs induced by the
extrastimuli, because a similar method has been recently
established for the evaluation of atrial vulnerability [24]. Atrial
burst pacing has been frequently used to induce AF. However,burst pacing sometimes induces nonspecific AF and atrial
tachycardia, even in control hearts. In fact, in our isolatedperfused heart, preliminary experiments showed induction ofnonspecific AF by atrial burst pacing in some of the control
hearts. That is why we applied an S3 extrastimuli protocol, in
which we confirmed that RARs were never induced in NTtreated control hearts. In AII-infused hearts, atrial extrastimuli
resulted in a high incidence of RAR, which were suppressed by

Fig. 7. Repetitive atrial response (RAR). (A) Two representative RAR observed in the AII-NT-HHR group. (B) Incidence of RAR. ∗P b 0.05, ∗∗P b 0.01, NS = notsignificant.

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O. Wakisaka et al. / Journal of Molecular and Cellular Cardiology 43 (2007) 616–626

pretreatment with HT. RARs were seen as rapid and irregularatrial excitations, mimicking AF. The electrophysiologicalmechanisms responsible for RAR are unclear. It has beenreported, however, that pre-existing nonuniformity in heterogeneous interstitial fibrosis can easily break the single activation
wavefront into multiple wavefronts [25]. Therefore, it issuggested that slowed and nonuniform conduction due toheterogeneous interstitial fibrosis may contribute to the formation of multiple wavefronts, leading to the occurrence of RAR.
Cardiac fibroblasts are the predominant secretory cells
producing extracellular matrix (ECM) proteins, key mediatorsof cardiac interstitial fibrosis [5]. This is why we used atrial
fibroblasts rather than atrial cardiomyocytes. In response to AII,ERK1/ERK2 phosphorylation and subsequent cellular proliferation occur via direct stimulation of the AT1 receptor [26–28].
Consistently, AII caused ERK1/ERK2 phosphorylation in
cultured atrial fibroblasts, which was attenuated by pretreatment
with HT. Activation of the AT1 receptor by AII is characterizedby conformational changes [19]. In contrast to candesartan, HT
did not reduce IP production, i.e. it did not induce conformational changes in the AT1 receptor in cultured fibroblasts in
response to AII, suggesting that HT might work below the levelof the AT1 receptor.
In in vivo experiments, the atrial tachypacing-induced model
has been established to represent clinical AF [29–33]. In some
reports, characteristic shortening of atrial ERP, induced by shortterm atrial tachypacing, was suppressed by pharmacologicalinhibition of the renin–angiotensin system [29,34]. Interestingly, in a canine AF model with rapid atrial pacing at 400 beats/
min for 5 weeks, rather than shortening the atrial ERP, extensive
interstitial fibrosis was found in the RA free wall in association
with a gradual conduction prolongation in the atria [4]. Because
long-term treatment with candesartan, which started 1 week
before the pacing and continued for 5 weeks during tachypacing,
prevented atrial fibrosis, Kumagai et al. [4] attributed theinteratrial conduction slowing to interstitial fibrosis, which led tothe initiation and maintenance of AF in their model, andconcluded that candesartan can prevent the promotion of AF bysuppressing the development of structural remodeling. When
compared with atrial tachypacing, our approach using continuous AII infusion without pacing may have some advantages,
because continuous AII infusion causes LA wall stretch(reflected as a slight increase in LVEDP) without heart failure,and causes atrial interstitial fibrosis without affecting atrial ERP.
These processes are more likely to underlie the mechanisms for
newly developed AF in patients with hypertension.
Besides the fibrotic signals evaluated in our experiments
using cultured fibroblasts, a large number of factors must be
involved in the AII-induced fibrosis observed in experiments invivo. For instance, cardiac function is of importance in thedevelopment of atrial fibrosis and AF [3]. In our continuous AII
infusion model, no overt heart failure was observed. No
difference in LV function was observed between the AII-HHR
and AII-HT groups, suggesting that improvement of LV functionis unlikely to be coupled to the preventive effects of HT on AF.
Regarding atrial function, it was impossible for us to directly
measure the hemodynamic parameters of the atrium. Alterna-

tively, we estimated the ANP mRNA expression levels of theLA, because stretch and/or AII have been demonstrated toincrease atrial ANP mRNA expression [35]. In the present study,
the ANP mRNA levels were increased in the AII-HHR group;
this effect was suppressed by the pretreatment of HT. It cantherefore be postulated that AII-induced LA pressure overload,
which was not reflected in LVEDP but in the LA ANP mRNAlevel, might be attenuated by HT treatment. However,substantial improvement of LV function, which did not appear
in our measured indices, might underlie the improvement of
atrial function. The ER is an organelle that participates in folding
secretory and membrane proteins, and ER chaperones are
upregulated in response to stress [36]. To investigate theinvolvement of ER stress in AII-induced fibrotic processes, weevaluated the levels of the major ER chaperones, GRP78 and
GRP94. The expression of both GRP78 and GRP94 proteins was
upregulated in response to AII. Pretreatment of HT, however, did
not influence the expression levels of these proteins. To ourknowledge, there have been no reports describing the role of ER
stress in atrial fibrosis induced by AII.
Information is limited with respect to the effects of inductionof the heat-shock response on AF substrate. Brundel et al. [13]reported that tachypacing applied to HL-1 (atrial myocytes)
induced myolysis, which was prevented by the overexpressionof HSP27 but not of HSP72. Because myolysis of atrial
myocytes is reported to be associated with the progression of
AF [37], the authors concluded that upregulation of HSPs,
particularly HSP27, protects tachypaced atrial myocytes frommyolysis, leading to a novel therapeutic approach in AF.
Subsequently, Brundel et al. [14], using HL-1 cells, reported
that atrial tachypacing-induced remodeling, including progressive reduction in the Ca2+ transient, L-type Ca2+ current, andaction potential duration in myocytes, was suppressed by HT or
GGA. They also demonstrated that the ERP shortening inducedby 1 week of atrial tachypacing in dogs was suppressed by HT
or GGA. By using the transfection technique in HL-1 cells,
Brundel et al. concluded that HSP27 rather than HSP72 plays a
predominant role in HT- or GGA-induced protection against
AF. There are distinctly different experimental approaches
between the studies of Brundel et al. [13,14] and our studies. In
experiments in vitro, while they evaluated myolysis [13] andelectrical remodeling [14] by introducing tachypacing to HL-1
myocytes, we evaluated the fibrotic signals evoked by direct
application of AII using isolated atrial fibroblasts. In addition, in
experiments in vivo, Brundel et al. did not evaluate structural
remodeling represented by atrial fibrosis. These differences inexperimental design may explain the disparity in the observations between our work, which shows that HSP72 induction isprotective, and that of Brundel et al., which showed thatinduction of HSP27 but not HSP72 is protective against AF.
Nevertheless, combining both sets of observations leads us toconclude that induction of the heat-shock response is expected
to be effective in preventing AF via prevention of both electrical
remodeling and structural remodeling.
There are several limitations to the present study. Firstly,
continuous infusion of AII caused marked elevation of arterial
BP. Therefore, we could not separate the direct effects of AII on

O. Wakisaka et al. / Journal of Molecular and Cellular Cardiology 43 (2007) 616–626

the fibroblasts that caused fibrotic change from the secondary
outcomes derived from increased BP. Secondly, although we
confirmed the suppressive effects of HSP72 on various fibrotic
signals in cultured fibroblasts by using siRNA targeting HSP72,
its inhibitory effects were incomplete. In addition, the effects of
HSP72 induction could not be isolated in experiments in vivo.
Besides the HSP family, HT is known to induce broad classes ofprotective proteins [7,38]. The anti-fibrotic effects of HT
observed in vivo, therefore, could not be solely explained by theeffects of HSP72 induction. Finally, although we evaluatedseveral fibrotic factors, including ERK1/2 phosphorylation, it is
impossible to directly connect these factors with antifibrotic
effects of HSP72 induction. Based on our observations, it isconceivable that HSP72 inhibits AII-induced ERK1/ERK2
phosphorylation, a critical upstream signal, subsequently
resulting in both prevention of fibroblasts to differentiate intomyofibroblast in association with an acquisition of α-SMA andinhibition of TGF-β1 secretion, collagen synthesis, and collagentype 1 expression. However, the possibility that HSP72 suppresses each of these fibrotic factors cannot be excluded.
In conclusion, our results suggest that the induction of theheat-shock response prevents atrial fibrosis and AF that are
mediated by the activation of the renin–angiotensin system, atleast partly through expression of HSP72. The mechanisms of
induction of the heat-shock response, either via HT or pharmacologic stimuli, may be effective in preventing atrial fibrosis
and AF where AII is involved.
Acknowledgments
This work was supported by grants-in-aid for scientificresearch from the Ministry of Education in Japan (grants
16590699, to Dr. Takahashi, and 17590756, to Dr. Saikawa).
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