Mitochondria are targets for
geranylgeranylacetone-induced cardioprotection against
ischemia-reperfusion in the rat heart

Tetsuji Shinohara, Naohiko Takahashi, Hiroaki Kohno, Kunitoshi Yamanaka, Tatsuhiko
Ooie, Osamu Wakisaka, Yukichi Murozono, Yayoi Taniguchi, Yasuko Torigoe, Masahide
Hara, Tatsuo Shimada, Tetsunori Saikawa and Hironobu Yoshimatsu
Am J Physiol Heart Circ Physiol 293:H1892-H1899, 2007. First published 22 June 2007;
doi: 10.1152/ajpheart.00493.2007
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Am J Physiol Heart Circ Physiol 293: H1892–H1899, 2007.
First published June 22, 2007; doi:10.1152/ajpheart.00493.2007.

Mitochondria are targets for geranylgeranylacetone-induced cardioprotection
against ischemia-reperfusion in the rat heart
Tetsuji Shinohara,1 Naohiko Takahashi,1 Hiroaki Kohno,1 Kunitoshi Yamanaka,1 Tatsuhiko Ooie,2Osamu Wakisaka,1 Yukichi Murozono,1 Yayoi Taniguchi,2 Yasuko Torigoe,2 Masahide Hara,1Tatsuo Shimada,3 Tetsunori Saikawa,2 and Hironobu Yoshimatsu1
1

Department of Internal Medicine 1, 2Department of Cardiovascular Science, Faculty of Medicine, and 3Department of
Health Science, School of Nursing, Oita University, Oita, Japan
Submitted 24 April 2007; accepted in final form 19 June 2007

cardioprotection against ischemia-reperfusion injury in rat
hearts (16, 25). Nevertheless, the precise mechanisms for
GGA-induced protection remain unknown. Since mitochondria
have been shown to be selective targets for heat stress (HS)induced cardioprotection (20), we hypothesized that the preservation of mitochondrial function could be involved in GGAor HS-induced cardioprotection against ischemia-reperfusion.
The mitochondrial ATP-sensitive potassium (mitoKATP)
channel is a putative channel in the inner mitochondrial
membrane (17). Although this channel has not yet beencloned, its pharmacological activation by nicorandil provides cardioprotection against ischemic insult or oxidative
stress and its blockade by 5-hydroxydecanoate (5-HD) or
glibenclamide prevents this protection (7, 24). It has also
been reported that opening of the mitoKATP channel has an
essential role in ischemic preconditioning-induced protection of mitochondria (6).
In the present study, using rat heart and rat neonatal cultured
cardiomyocytes, we investigated the mechanism by which
HSP72 expression induces cardioprotection. In experiments
using isolated perfused hearts, we investigated the effects ofeither 5-HD or glibenclamide on left ventricular (LV) functional recovery and creatine kinase (CK) release during a
30-min reperfusion following 20 min of no-flow global ischemia. At the end of the reperfusion period, the mitochondria
were isolated for evaluation of respiratory function and theirultrastructure. In experiments using cultured cardiomyocytes,
the effect of 5-HD on GGA-induced cardioprotective effects
was investigated using small-interfering RNA (siRNA) thattargets HSP72 because GGA-induced cardioprotective effects
may involve HSP72 expression.
MATERIALS AND METHODSCARDIOMYOCYTES POSSESS ABUNDANT mitochondria, which are
key organelles involved in ischemia-reperfusion injury (14,19). Damage to mitochondria causes reduced respiratory function and ATP synthesis, resulting in irreversible cardiomyocyte
damage (14, 19). Our laboratory has previously reported that
geranylgeranylacetone (GGA), an anti-ulcer agent, induces
expression of heat shock protein 72 (HSP72) and provides

All experimental procedures were approved by and performed inaccordance with the guidelines for the care and use of laboratory
animals of the Physiological Society of Oita University, Japan.
Materials. Monoclonal immunoglobulin G (IgG) cross-reactive to
inducible HSP72 antibody was obtained from Stressgen Biotechnologies (Victoria, BC, Canada). Horseradish peroxidase-linked F(ab⬘)2
fragment from sheep anti-mouse immunoglobulin and reagents forWestern blot assay and enhanced chemiluminescence were purchasedfrom Amersham (Piscataway, NJ). Bradford protein assay kits werepurchased from Bio-Rad (Richmond, CA). 5-HD and glibenclamide werepurchased from ICN Biomedicals (Costa Mesa, CA) and Wako (Osaka,
Japan), respectively. GGA was provided by Eisai (Tokyo, Japan).

Address for reprint requests and other correspondence: T. Shinohara, Dept.
of Internal Medicine 1, Faculty of Medicine, Oita Univ., 1-1 Idaigaoka,
Hasama, Oita 879-5593, Japan (e-mail: shinohar@med.oita-u.ac.jp).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

heat shock protein 72; mitochondrial ATP-sensitive potassium channel; 5-hydroxydecanoate; glibenclamide

H18920363-6135/07 $8.00 Copyright © 2007 the American Physiological Society

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Shinohara T, Takahashi N, Kohno H, Yamanaka K, Ooie T,
Wakisaka O, Murozono Y, Taniguchi Y, Torigoe Y, Hara M,
Shimada T, Saikawa T, Yoshimatsu H. Mitochondria are targets
for geranylgeranylacetone-induced cardioprotection against ischemia-reperfusion in the rat heart. Am J Physiol Heart Circ
Physiol 293: H1892–H1899, 2007. First published June 22, 2007;
doi:10.1152/ajpheart.00493.2007.—It has been shown that orally
administered geranylgeranylacetone (GGA), an anti-ulcer drug, induces expression of heat shock protein 72 (HSP72) and provides
protection against ischemia-reperfusion in rat hearts. The underlying
protective mechanisms, however, remain unknown. Mitochondria
have been shown to be a selective target for heat stress-induced
cardioprotection. Therefore, we hypothesized that preservation of
mitochondrial function, owing to an opening of a putative channel inthe inner mitochondrial membrane, the mitochondrial ATP-sensitive
potassium (mitoKATP) channel, could be involved in GGA- or heatstress-induced cardioprotection against ischemia-reperfusion. Rats
were treated with oral GGA or vehicle. Twenty-four hours later, eachheart was isolated and perfused with a Langendorff apparatus. GGAtreated hearts showed better functional recovery, and less creatine
kinase was released during a 30-min reperfusion period, after 20 minof no-flow ischemia. Concomitant perfusion with 5-hydroxydecanoate(5-HD, 100 ␮M) or glibenclamide (10 ␮M) abolished the GGAinduced cardioprotective effect. GGA also showed preserved mitochondrial respiratory function, isolated at the end of the reperfusion
period, which was abolished with 5-HD treatment. GGA prevented
destruction of the mitochondrial structure by ischemia-reperfusion, as
shown by electron microscopy. In cultured cardiomyocytes, GGA
induced HSP72 expression and resulted in less damage to cells,
including less apoptosis in response to hypoxia-reoxygenation. Treatment with 5-HD abolished the GGA-induced cardioprotective effects
but did not affect HSP72 expression. Our results indicate that preserved mitochondrial respiratory function, owing to GGA-induced
HSP72 expression, may, at least in part, have a role in cardioprotection against ischemia-reperfusion. These processes may involve opening of the mitoKATP channel.

MITOCHONDRIA AND GGA-INDUCED CARDIOPROTECTION

Fig. 1. Experimental protocol. A: isolated perfused heart experiments.
B: experiments using cultured cardiomyocytes. Control (CNT) group, rats
treated with oral (po) vehicle; geranylgeranylacetone (GGA) group, rats treatedwith oral GGA (200 mg/kg); heat stress (HS) group, rats treated with HS at
43°C for 15 min; 5-HD, 5-hydroxydecanoate; Glib, glibenclamide; LV, left
ventricular; CK, creatine kinase; HSP72, heat shock protein 72; LDH, lactate
dehydrogenase; TUNEL, transferase-mediated dUTP nick-end labeling. See
text for details.

recorder (WS-681G, Nihon Kohden, Tokyo, Japan) and stored on a
PCM data recorder (RD-111T, TEAC, Tokyo, Japan) for later analysis.
Western blot analysis. At the end of the reperfusion period, hearts
were rapidly removed from the Langendorff apparatus and frozen inliquid nitrogen. Western blot analysis was performed as previously
described (16, 25). To analyze the expression of HSP72, frozen heartsamples were homogenized with SDS sample buffer (20% glycerol,
6% SDS, and 0.12 M Tris, pH 6.8), centrifuged, and boiled. The total
protein concentration of each heart was quantified by the Bradford
method (4). The preparations were diluted in dissociation buffer. An
equal amount of total protein from each fraction was electrophoresedby 8.5% SDS-PAGE and transferred electrophoretically to a polyvinylidene fluoride membrane. After being transferred and blocked with
0.5% nonfat milk, the membranes were incubated with antibodies.
The proteins were detected by enhanced chemiluminescence with
exposure to Hyperfilm. The amount of protein on the immunoblots
was quantified using National Institutes of Health (Bethesda, MD)
image analysis software.
Mitochondria isolation. At the end of the reperfusion period,
mitochondria were isolated from the hearts by differential centrifugation (2, 22). Briefly, hearts were dissected and immersed in ice-cold
medium containing (in mM) 225 mannitol, 75 sucrose, 10 Tris, and 2
EGTA, and 0.1% bovine serum albumin (BSA) (pH 7.2 at 4°C). Afterthe atria and major vessels were discarded, ventricles were washed in
the ice-cold medium, minced, and homogenized. The homogenate wascentrifuged at 800 g for 10 min at 4°C. The supernatant was retrieved
and centrifuged at 7,000 g for 10 min. The resulting pellet was thenresuspended in the buffer (as described above, but without EGTA) andrecentrifuged at 7,000 g for 10 min. The final pellet was resuspended
in the EGTA-free buffer and kept on ice.
Mitochondrial respiration. Mitochondrial oxygen consumption
was measured as previously reported (2, 22) using a Clark-type
oxygen electrode connected to an oxygraph (Central Kagaku, Tokyo,
Japan). Mitochondria (⬃1 mg of protein) and 0.25 mg of fat-free BSA
were placed in 1 ml of respiration medium containing (in mM) 100
KCl, 75 mannitol, 25 sucrose, 0.05 EDTA (dipotassium salt), 10
Tris 䡠 HCl, and 10 KH2PO4-Tris (pH 7.4) and stirred at 30°C. State 4
respiration was initiated using 5 mM glutamate and 5 mM malate or
5 mM succinate and 1 ␮M rotenone. State 3 respiration was initiatedby the addition of ADP to the respiration medium. The respiratory
control index (RCI) was calculated as the ratio of the state 3 to state
4 mitochondrial oxygen consumption rates.
Electron microscopic findings. Isolated mitochondria were fixed incacodylate-buffered (pH 7.4) 2.5% glutaraldehyde and paraformaldehyde at 4°C for 2 h and then fixed in cacodylate-buffered (pH 7.4) 2%
osmium tetroxide and 0.5% potassium ferrocyanide at 4°C for 2 h.
The preparations were dehydrated in a graded series of ethanol andembedded in epoxy resin. The preparations were then stained withuranyl acetate and lead cystate and viewed under a transmission
electron microscope (JEM-100CX, JOEL, Tokyo, Japan).
GGA-induced HSP72 expression and hypoxia-reoxygenation in
cardiomyocytes. Neonatal cardiomyocytes were prepared from 3-dayold Wistar rats, as described previously (23). Briefly, cardiomyocytes
were placed on 35-mm culture dishes at a density of 5 ⫻ 106 cells perdish and cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum at 37°C and under amixture of 5% CO2-95% room air. On day 3, the medium was
replaced with serum-free DMEM supplemented with 5 ␮g/ml transferrin, and the cells were cultured for 24 h. GGA supplemented with
tocopherol was dissolved in absolute ethanol (final concentration,⬍0.1%). Our preliminary experiments investigating dose-dependentchanges in HSP72 expression revealed that treatment with 10⫺5 M
GGA induced maximal HSP72 expression (Fig. 6A). Furthermore,experiments estimating time-dependent changes in HSP72 expression
found that GGA-induced HSP72 expression reached a peak at 4 h(Fig. 6B). According to these preliminary results, HSP72 expression

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Animals. Male Sprague-Dawley rats (200 –250 g) were used. GGA,
as an emulsion with 5% gum arabic and 0.008% tocopherol, wasgiven orally at a dose of 200 mg/kg (16, 25). Rats were classified into
three groups. The control group was treated with a single oral
administration of vehicle. The GGA group received a single oral
administration of GGA. The HS group was treated in a water bath at
43°C for 15 min (16). Rectal temperature was monitored for confirmation of body temperature elevation. Twenty-four hours after treatment, rats were anesthetized (pentobarbital sodium, 50 mg/kg ip) andheparinized (500 IU/kg ip). Hearts were then isolated and prepared forexperiments (Fig. 1A).
Isolated perfused heart experiments. Each heart was isolated andperfused retrogradely using the Langendorff method, with KrebsHenseleit buffer (pH 7.4) containing (in mM) 118 NaCl, 4.7 KCl, 2.5
CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, and 11.0 glucose,equilibrated with 95% O2-5% CO2 gas mixture at 36.5°C at a constantpressure of 75 mmHg (16, 25). A water-filled latex balloon wasinserted through the mitral orifice into the LV, and the LV enddiastolic pressure (LVEDP) was adjusted to 0 –5 mmHg. The heart
was covered with water-jacketed glassware, and the relative humidity
was maintained for at least 90%. The perfusion protocol is shown in
Fig. 1. Each heart from the three groups was perfused in the presenceor absence of 5-HD (100 ␮mol/l) or glibenclamide (10 ␮mol/l).
Concomitant perfusion with 5-HD or glibenclamide was initiated 10
min before the introduction of ischemia and continued to the end of
the reperfusion period. The hearts were subjected to normothermic
no-flow global ischemia for 20 min, followed by reperfusion for 30
min (16, 25). The coronary effluent during the 30-min reperfusion
period was collected to measure CK release. LV pressure was monitored using a pressure transducer to record the peak positive andnegative first derivatives of LV pressure (dP/dtmax and dP/dtmin,
respectively). LV developed pressure (LVDP) was defined as thedifference between the LV systolic and diastolic pressures. Coronary
perfusion pressure (CPP) was defined as the hydraulic pressure
measured at the level of aortic cannulation. LV pressure, CPP, andelectrocardiograms were continuously recorded using a polygraph

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MITOCHONDRIA AND GGA-INDUCED CARDIOPROTECTION

Table 1. Hemodynamic parameters at baseline

CNT
GGA
HS
CNT-5-HD
GGA-5-HD
HS-5-HD
CNT
GGAHS
CNT-Glib
GGA-Glib
HS-Glibn

LVDP, mmHg

HR, beats/min

CPP, mmHg

dP/dtmax, mmHg/s

dP/dtmin, mmHg/s

8
9
9
8
8
8
7
7
7
7
7
7

104⫾6
96⫾13
96⫾6
102⫾13
104⫾6
99⫾8
103⫾5
101⫾4
100⫾5
101⫾5
101⫾7
103⫾5

346⫾18325⫾9331⫾16324⫾17324⫾11
342⫾22341⫾17321⫾15338⫾15321⫾11
341⫾18327⫾26

81⫾2
80⫾3
84⫾3
84⫾4
81⫾4
82⫾3
79⫾4
79⫾2
80⫾6
81⫾5
83⫾5
81⫾5

3,170⫾172
3,270⫾114
3,161⫾97
3,108⫾145
3,189⫾120
3,050⫾125
3,088⫾82
3,287⫾156
3,269⫾101
3,167⫾72
3,085⫾164
2,987⫾173⫺2,768⫾83⫺2,801⫾112⫺2,762⫾51⫺2,808⫾152⫺2,924⫾156⫺2,781⫾170⫺2,807⫾160⫺2,844⫾112⫺2,725⫾128⫺2,840⫾126⫺2,772⫾155⫺2,745⫾170

was evaluated after 4 h with treatment of 10⫺5 M GGA. To evaluate
the effect of 5-HD on GGA treatment, the cultured myocytes weretreated with 10⫺5 M GGA or vehicle for 4 h in the culture medium,and 5-HD (500 ␮M) was added 10 min before hypoxia was induced.
Some cardiomyocytes were lysed for Western blot analysis. To
evaluate the tolerance to hypoxia-reoxygenation, the remaining cardiomyocytes were incubated in serum-free DMEM without glucose
under a hypoxic gas mixture (95% N2-5% CO2) at 37°C for 3 h,followed by reoxygenation under a normoxic gas mixture for 1 h. The
supernatant was carefully collected for lactate dehydrogenase (LDH)
determination using an LDH assay kit (Eiken Chemical, Tokyo,
Japan). For the apoptosis assay, cells were fixed after reoxygenation in
4% polyparaformaldehyde solution in 0.1 M NaH2PO4 and permeabilized with 0.5% Triton X-100 (Sigma, St. Louis, MO). Apoptotic
cells were detected with the in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method using an

Fig. 2. A and B: effects of 5-HD (A) and
Glib (B) on serial changes of LVDP andpeak positive and negative first derivativesof LV pressure (dP/dtmax and dP/dtmin, respectively) during the experimental period.
Twenty minutes of no-flow global ischemia
was followed by 30 min of reperfusion. Data
are means ⫾ SE; n ⫽ 8 or 9 hearts/group.
*P ⬍ 0.05 and **P ⬍ 0.01 vs. corresponding 5-HD- or Grib-perfused groups. C and
D: effects of perfusion with 5-HD (C) or
Glib (D) on GGA- or HS-induced reductionin amount of CK released during the 30-min
reperfusion period. Amount of released CK
is expressed relative to ventricular weight.
Data are means ⫾ SE. **P ⬍ 0.01 vs.
control group.

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Values are means ⫾ SE; n, number of hearts. LVDP, left ventricular developed pressure; HR, heart rate; CPP, coronary perfusion pressure; dP/dtmax anddP/dtmin, peak positive and negative first derivatives of LV pressure, respectively; CNT, control; GGA, geranylgeranylacetone; HS, heat shock; 5-HD,
5-hydroxydecanoate; Glib, glibenclamide.

MITOCHONDRIA AND GGA-INDUCED CARDIOPROTECTION

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control cells were transfected with negative control siRNA. Twentyfour hours after transfection, cells were incubated with GGA orvehicle for 4 h. HSP72 expression was evaluated, and hypoxiareoxygenation was performed.
Statistical analysis. Data are expressed as means ⫾ SE. Serialchanges in LVDP, LVEDP, CPP, heart rate, and dP/dt were analyzed
by two-way ANOVA and by the Bonferroni/Dunn test, unless otherwise specified. The hemodynamic parameters at baseline, the ratio of
released CK to ventricular weight, the LDH content in the cultured
medium, and the percentage of apoptotic cells were compared amonggroups by one-way ANOVA followed by the Bonferroni/Dunn test.
The relative intensity of each protein and RCI were compared using
the Mann-Whitney U-test. A value of P ⬍ 0.05 was consideredsignificant.

Isolated perfused heart experiments. As shown in Table 1,
LVDP, heart rate, CPP, and dP/dt did not differ among the 12groups at baseline. Figure 2, A and B, shows the serial changes
in LVDP and dP/dt during the experimental period. The recovery of LVDP and dP/dt with reperfusion was greater in the
GGA and HS groups compared with the control group (P ⬍0.05 for each by ANOVA). Perfusion with 5-HD abolished theimproved functional recovery observed in the GGA- and HStreated hearts (Fig. 2A). Heart rate and CPP did not differ
significantly among the 12 groups at any period (data notshown). Perfusion with glibenclamide had the same effect as
5-HD (Fig. 2B). The amount of CK released during the 30-min

Fig. 3. Effects of 5-HD perfusion on GGA- or HS-induced HSP72 expression.
A: HSP72 expression without 5-HD perfusion. B: HSP72 expression with
5-HD perfusion. Hearts were isolated at the end of the reperfusion period andprepared for Western blot analysis. Representative immunoblots and relative
band densities of cardiac HSP72 are shown. Data are means ⫾ SE; n ⫽ 7hearts/group. *P ⬍ 0.05 and **P ⬍ 0.01 vs. control group. †P ⬍ 0.05 vs. GGA
group.

apoptosis kit (Medical Biological Lab, Nagoya, Japan). Slides weretreated with a mixture of terminal deoxynucleotidyl tranferase, FITCdUTP, and TdT buffer II at 37°C for 1 h. After being washed with
PBS, the cells were mounted with mounting medium of 4⬘,6-diamidino-2-phenylindole.
siRNA transfection. The siRNAs targeting HSP72 were obtained
from Ambion (Austin, TX) and transfected into cells at a concentration of 10 nM with siPORT NeoFX Transfection (Ambion) (23). The

Fig. 4. Respiratory control index (RCI). RCI was calculated as the ratio of thestate 3 to state 4 mitochondrial oxygen consumption rates. Mitochondria wereisolated at the end of the reperfusion period. NAD⫹- (A) and FAD-linked
respiration (B) is shown. Data are means ⫾ SE; n ⫽ 7 hearts/group. *P ⬍ 0.05
vs. control group.

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MITOCHONDRIA AND GGA-INDUCED CARDIOPROTECTION

in cultured cardiomyocytes. When assessed 4 h after GGA
application, a GGA concentration of 10⫺5 M induced thegreatest HSP72 expression level (Fig. 6A). HSP72 expression
reached a maximum level 4 h after application of 10⫺5 M GGA(Fig. 6B).
The effect of 5-HD against GGA-induced HSP72 expression
in cultured rat neonatal cardiomyocytes. Figure 6C shows
representative immunoblots and relative band densities of
HSP72 in cultured rat neonatal cardiomyocytes. Irrespective of
the application of 5-HD, cardiomyocytes treated with GGA
showed increased HSP72 expression compared with that incontrol cardiomyocytes (P ⬍ 0.01).
The effects of GGA and 5-HD treatment on hypoxia-reoxygenation injury in cultured rat neonatal cardiomyocytes.
Figure 7, A and B, shows representative photomicrographs of
TUNEL staining and the percentages of apoptotic cells in
cultured cardiomyocytes after hypoxia-reoxygenation. GGA
treatment reduced the number of TUNEL-positive cells(18.2 ⫾ 2.0% vs. 7.4 ⫾ 1.1%, P ⬍ 0.01). However, concomitant application of 5-HD abolished the protective effects of
GGA (16.2 ⫾ 1.7% vs. 7.4 ⫾ 1.1%, P ⬍ 0.01). GGA treatment
reduced the level of LDH release in response to hypoxiareoxygenation compared with vehicle treatment (0.016 ⫾0.005 vs. 0.050 ⫾ 0.004 IU/ml, P ⬍ 0.01) (Fig. 7C).
However, a concomitant application of 5-HD abolished theprotective effects of GGA (0.042 ⫾ 0.006 vs. 0.016 ⫾ 0.005
IU/ml, P ⬍ 0.01).
The effect of HSP72-siRNA on HSP72 expression and hypoxia-reoxygenation injury. As shown in Fig. 8A, the HSP72targeted siRNA effectively suppressed the GGA-induced

Fig. 5. Electron microscope results. In isolated mitochondria from the control group, most of the inner andouter membranes were disrupted, displaying destroyed
cristae formation and a swollen matrix. These alterations were reduced, however, in isolated mitochondria
from the GGA or HS group, although some mitochondria were still markedly damaged.

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reperfusion was lower in the GGA and HS groups than in thecontrol group (P ⬍ 0.01) (Fig. 2, C and D). Perfusion with
5-HD abolished the reduction in CK release observed in the
GGA and HS groups (Fig. 2C). Perfusion with glibenclamide
had the same effect as 5-HD (Fig. 2D).
GGA-induced cardiac HSP72 expression. Figure 3, A and B,
shows the cardiac HSP72 expression level in the hearts at theend of reperfusion. Irrespective of perfusion with 5-HD, thehearts treated with GGA or HS showed increased HSP72
expression levels compared with control hearts (P ⬍ 0.05 andP ⬍ 0.01, respectively). The HSP72 expression level inHS-treated hearts was greater than that in GGA-treatedhearts (P ⬍ 0.05).
Mitochondrial respiration. Figure 4, A and B, shows the
respiratory function of mitochondria at the end of the reperfusion period. The RCIs for malate (complex I substrate)- andsuccinate (complex II substrate)-energized mitochondria wereincreased in the GGA and HS groups compared with thecontrol group (P ⬍ 0.05 for each). Concomitant perfusion with
5-HD abolished the improved respiration function of mitochondria in GGA- and HS-treated hearts.
Electron microscopic findings. In mitochondria isolated
from the control group, most of the inner and outer membranes
were disrupted, displaying disrupted cristae formation and aswollen matrix (Fig. 5). These alterations were reduced, however, in mitochondria isolated from the GGA and HS groups,
although some mitochondria were substantially damaged.
GGA-induced HSP72 expression in cultured rat neonatal
cardiomyocytes. Figure 6, A and B, shows the effects ofconcentration- and time-dependent GGA induction of HSP72

MITOCHONDRIA AND GGA-INDUCED CARDIOPROTECTION

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Fig. 7. Effects of GGA and 5-HD treatment on hypoxia-reoxygenation injury
in cultured rat neonatal cardiomyocytes. A: representative photomicrographs of
TUNEL staining in cultured cardiomyocytes after hypoxia-reoxygenation.
Nuclei with green staining indicate TUNEL-positive cells. B: quantitative
results of TUNEL staining for different groups. C: amount of LDH released in
response to hypoxia-reoxygenation. DAPI, 4⬘,6-diamidino-2-phenylindole.
Five independent cultures were evaluated. Data are means ⫾ SE; **P ⬍ 0.01
vs. vehicle group; ††P ⬍ 0.01 vs. GGA-treated cells in the absence of 5-HD.

Fig. 6. A and B: representative immunoblots and relative band densities of
GGA-induced HSP72 expression in cultured cardiomyocytes according to
GGA dose and incubation time. C: effect of 5-HD on GGA-induced HSP72
expression in cultured rat neonatal cardiomyocytes. Data are means ⫾ SE; n ⫽5 independent cultures/group. **P ⬍ 0.01 vs. vehicle group.

HSP72 expression in the presence or absence of 5-HD. In
GGA-treated cardiomyocytes, the LDH release in response tohypoxia-reoxygenation was increased with 5-HD treatment(0.012 ⫾ 0.002 vs. 0.030 ⫾ 0.004 IU/ml, P ⬍ 0.01) (Fig. 8B).
In the absence of 5-HD, treatment with HSP72-targeted siRNA

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MITOCHONDRIA AND GGA-INDUCED CARDIOPROTECTION

inhibited the GGA-induced protective effects on LDH release(0.012 ⫾ 0.002 vs. 0.038 ⫾ 0.008 IU/ml, P ⬍ 0.01). Concomitant treatment with 5-HD did not affect the amounts of LDH
released (0.030 ⫾ 0.004 vs. 0.034 ⫾ 0.006 IU/ml, not significant).
DISCUSSION

In the present study, we demonstrated that rat hearts treatedwith GGA or HS were protected from ischemia-reperfusion
injury. This was shown by better LV function recovery andreduced CK release during reperfusion. Preservation of mitochondrial respiratory function and structure was also demonstrated, when assessed at the end of reperfusion. These observations indicate that upregulation of HSP72 with GGA or HS
protects the mitochondria from ischemia-reperfusion injury,
resulting in improved preservation of cardiac function.
Regarding the role of mitochondria as a target for theprotective effects of HS, Polla et al. (20), using cultured cells,
reported that treatment with HS prevented hydrogen peroxideinduced changes in mitochondrial membrane potential andcristae formation and that the protective effects of HS were
well correlated with the expression of HSP72. Jayakumar et al.
(15) reported that rat hearts transfected with the HSP72 gene

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Fig. 8. Effect of HSP72-specific small-interfering (siRNA) on HSP72 expression and hypoxia-reoxygenation injury in cultured cardiomyocytes. A: representative immunoblots and relative band densities of HSP72. B: amount of
LDH release in response to hypoxia-reoxygenation. Data are means ⫾ SE; n ⫽5 independent cultures/group. **P ⬍ 0.01 vs. GGA-treated cells in the absenceof 5-HD and HSP72-specific siRNA.

via intracoronary infusion showed protection of mitochondrial
respiratory function after ischemia-reperfusion, which was associated with improved preservation of ventricular function.
They suggested that one of the mechanisms for HSP72-mediated cardioprotection may be the opening of the mitoKATP
channel, although no evidence was shown (15). The importantrole of the mitoKATP channel in myocardial ischemic tolerance
after HS treatment has since been reported in animal studies,both in vitro and in vivo (1, 12, 18). In isolated perfused
rat hearts, preperfusion with either 10 ␮M glibenclamide or
100 ␮M 5-HD abolished the reduction in regional ischemiainduced infarct size produced by whole body HS treatment
24 h before ischemia (12). With the use of an in vivo rabbit
model of acute myocardial infarction, the reduction in infarct
size with HS treatment 24 h before coronary occlusion was
abolished with intravenous 0.3 mg/kg glibenclamide or 5
mg/kg 5-HD administered 10 min before coronary occlusion(18). These studies, however, did not evaluate mitochondrial
respiratory function in response to ischemia-reperfusion (1, 12,18). In our perfused rat heart experiment, concomitant perfusion with 100 ␮M 5-HD or 10 ␮M glibenclamide abolished the
GGA- or HS-induced improvement in LV functional recovery
during reperfusion. Furthermore, GGA- or HS-treated hearts
showed preserved mitochondrial respiratory function, withpreservation of mitochondrial ultrastructure, when assessed at
the end of the reperfusion period. The preservation of mitochondrial respiratory function with GGA or HS was abolishedby treatment with 100 ␮M 5-HD. The pharmacological selectivity of these widely used mitoKATP-channel blockers, at theconventional inhibitory concentrations (1, 12, 18) used in thepresent study, may indicate the important role of the mitoKATP
channel in GGA- or HS-induced preservation of mitochondrial
respiratory function, leading to cardioprotection against ischemia-reperfusion.
The role of the mitoKATP channel in modulating cardiac
mitochondrial function has been investigated previously (6,13). Using isolated mitochondria from rat ventricles, Holmuhamedov et al. (13) demonstrated that the pharmacologicalopening of mitoKATP channel induced rapid membrane depolarization, a decrease in the rate of ATP synthesis, and a
resultant compensatory increase in the rate of mitochondrial
respiration. In addition to energy production, mitochondria canstore Ca2⫹, a function that is critical for the maintenance of
cellular Ca2⫹ homeostasis (5). The mitoKATP channel opening
was also shown to prevent cellular Ca2⫹ overload by accumulating and retaining Ca2⫹ in the matrix (13). These mechanisms may underlie the cardioprotection induced by GGA orHS, as demonstrated in the present study. Further studies areneeded to show how enhanced expression of HSP72, induced
by GGA or HS, modulates the activity of the mitoKATP
channel. As shown in Fig. 3, at the end of the reperfusion
period, HSP72 was abundantly expressed in GGA- or HStreated heart, even in those concomitantly perfused with 5-HD.
Consistent with this finding, the study of Hoag et al. (12)reported that the enhanced expression of HSP72 in HS-treatedrabbit heart was not reduced by preperfusion with glibenclamide or 5-HD. Taken together, these results indicate that
HSP72 degradation does not underlie the ability of 5-HD toabolish cardioprotection.
Until now, we have attributed the opening of the mitoKATP
channel, and the resultant preservation of mitochondrial func-

MITOCHONDRIA AND GGA-INDUCED CARDIOPROTECTIONGRANTSThis study was supported partly by Japanese Ministry of Education,
Science, and Culture Grants-in-Aid 16590699 (to N. Takahashi) and 17590756(T. Shinohara).
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tion, to HSP72-induced cardioprotection by GGA or HS. However, another mechanism may be involved. Following ischemia-reperfusion, cardiomyocytes die, by apoptosis as well as
necrosis, and the anti-apoptotic effect of HSP72 in HS-treatedcells has been reported to be related to suppression of the stresskinase c-Jun NH2-terminal kinase (JNK) (8). A more recent
study demonstrated that HSP72 downregulates JNK by accelerating its dephosphorylation, which reduces the susceptibilityof myogenic cells to simulated ischemia-reperfusion (9). However, HSP72 essentially acts as a molecular chaperone (3).
HSP72 supports the transport of newly synthesized mitochondrial proteins, including respiratory chain enzymes, into mitochondria. The well-preserved enzyme activity of the mitochondrial respiratory chain may be due to the enhanced RCI after
ischemia-reperfusion observed in mitochondria isolated from
GGA- or HS-treated hearts.
Although we confirmed the protective effects of HSP72
expression in cultured cardiomyocytes using siRNA that targets HSP72, the effects of HSP72 induction could not be
isolated in experiments in vivo. In addition to the HSP family,
GGA is known to induce expression of many classes ofprotective proteins (11, 21). Therefore, the protective effects of
GGA observed in experiments in vivo could not be solely
explained by the effects of HSP72. In this regard, effective
elimination of HSP72 expression by HSP72-specific siRNA
was observed in vitro, together with the elimination of protection against hypoxia-reoxygenation. These results strongly
support the validity of our hypothesis that GGA-induced
HSP72 expression provides cardioprotection.
There are several limitations in the present study. First, weevaluated the effects of HSP72-siRNA only in experiments thatinvestigated hypoxia-reoxygenation injury, i.e., LDH release.
Future studies will be required to investigate the effects of
HSP72-siRNA on mitoKATP channel effectors as RCI. Second,although 5-HD is generally regarded as a specific inhibitor of
mitoKATP channel, it has been clarified that 5-HD has multiple
actions independent of mitoKATP channel (10). Thus alternative mechanisms should be taken into consideration as interpreting our results using 5-HD.
In conclusion, our observations indicate that the preserved
mitochondrial respiratory function with GGA-induced HSP72
expression may, at least in part, have a role in cardioprotection
against ischemia-reperfusion. An opening of the mitoKATP
channel may be involved in these processes.

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