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Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0529
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Mitochondrial membrane potential (
m) is
severely compromised in the myocardium after
ischemia-reperfusion and triggers apoptotic events leading
to cell demise. This study tests the hypothesis that mitochondrial
ATP-sensitive K+ (mitoKATP) channel activation
prevents the collapse of m in myocytes during
anoxia-reoxygenation (A-R) and is responsible for cell protection via
inhibition of apoptosis. After 3-h anoxia and 2-h
reoxygenation, the cultured myocytes underwent extensive damage, as
evidenced by decreased cell viability, compromised membrane
permeability, increased apoptosis, and decreased ATP concentration. Mitochondria in A-R myocytes were swollen and fuzzy as
shown after staining with Mito Tracker Orange CMTMRos and in an
electron microscope and exhibited a collapsed
m, as monitored by
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Cytochrome c was released from mitochondria
into the cytosol as demonstrated by cytochrome c
immunostaining. Activation of mitoKATP channel with
diazoxide (100 µmol/l) resulted in a significant protection against
mitochondrial damage, ATP depletion, cytochrome c loss, and
stabilized m. This protection was blocked by
5-hydroxydecanoate (500 µmol/l), a mitoKATP
channel-selective inhibitor, but not by HMR-1098 (30 µmol/l), a
putative sarcolemmal KATP channel-selective inhibitor.
Dissipation of m also leads to opening of
mitochondrial permeability transition pore, which was prevented by
cyclosporin A. The data support the hypothesis that A-R disrupts
m and induces apoptosis, which are prevented by the activation of the mitoKATP channel. This further
emphasizes the therapeutic significance of mitoKATP channel
agonists in the prevention of ischemia-reperfusion cell injury.
apoptosis; myocytes; ATP; permeability transition pore; cytochrome c
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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MITOCHONDRIA ARE MAJOR MYOCYTE organelles, and they play an important role in cell life and death. It is well known that myocardial ischemia-reperfusion (I/R) induces significant pathological changes in mitochondria (17). The impaired mitochondrial function after I/R is due to imbalance of cytosolic ions, electron transport, production of free radicals, and alteration of membrane potential (15, 28, 32), eventually leading to apoptosis in the ischemic myocardium (2, 5, 29).
Mitochondrial membrane potential (m) originates from
the asymmetric distribution of protons across the inner mitochondrial membrane and is essential for the maintenance of mitochondrial function. The relationship between m and pathological
conditions such as anoxia and apoptosis was the topic of
several recent studies (8, 18, 20, 37).
m is compromised due to the opening of permeability
transition pores at an early stage of apoptosis, whereas
m is needed for mitochondrial ATP production during apoptosis (36).
It has been reported (11, 22, 34, 35) that ATP-sensitive
K+ (KATP) channel openers exert
cardioprotective effects in various animal models of
ischemia-reperfusion. According to these studies, the mitochondrial
KATP(mitoKATP) channel-selective agonist
diazoxide improved postischemic functional recovery in isolated
rabbit and rat hearts (11, 34). It has been indicated
that diazoxide attenuated I/R injury due to preservation of
mitochondrial function (16). 5-Hydroxtdecanoate (5-HD), a
mitoKATP channel blocker, blocked
cardioprotection by diazoxide (11, 35). Moreover, mitoKATP openers depolarized mitochondrial membrane
potential by 10 mV (13). It is likely that the mechanism
of cardioprotection against ischemic injury by
mitoKATP channel may involve stabilization of
m. To address this question, the effect of mitoKATP channel on m as well as
cytochrome c release and apoptosis was investigated.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Diazoxide, mouse monoclonal anti-cytochrome c, and fluorecein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (Fab fragment) were purchased from Sigma (St. Louis, MO). 5-HD was purchased from ICN Biomedical (Costa Mesa, CA). Cyclosporin A was purchased from Biomolecular Research Labs (Plymouth Meeting, PA). HMR-1098 was a gift from Dr. Garrett Gross (Milwaukee, WI). All fluorescent dyes were purchased from Molecular Probes (Eugene, OR).
Experimental Protocols
Primary myocyte-rich cultures of the neonatal rat myocytes were prepared as described previously (36). Briefly, ventricles from hearts of 1- to 2-day-old rats were dissociated with trypsin and collagenase. The cells were resuspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 U/ml each of penicillin and streptomycin. To selectively enrich the myocytes, dissociated cells were preplated for 2 h to allow nonmyocytes to attach to the bottom of the culture dish. The resultant suspension of myocytes was transferred onto collagen-coated 60-mm or 100-mm culture dishes. Bromodeoxyuridine (100 µM) was added during the first 24-36 h to prevent proliferation of nonmyocytes. The experiments were performed on day 3 of culture, and myocytes were divided into the following six groups (Fig. 1).
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Group 1: control. The myocytes were incubated in Tyrode solution with glucose (25 mmol/l) during the entire experimental period.
Group 2: A-R. To induce complete anoxia, Tyrode solution was deoxygenated by bubbling with purified nitrogen for 1 h before the experiments. Myocytes were exposed to anaerobic glucose-free Tyrode solution and placed into the anoxic chamber (Forma 1025 anaerobic system) for 3 h of anoxia and then kept in normal Tyrode solution and returned to the CO2 incubator for 2 h of reoxygenation.
Group 3: diazoxide + A-R. The myocytes were preincubated with diazoxide (100 µmol/l) for 20 min before A-R.
Group 4: 5-HD + diazoxide + A-R. The myocytes were preincubated first with 5-HD (500 µmol/l) for 10 min and then with 5-HD and diazoxide for 20 min before A-R.
Group 5: HMR-1098 + diazoxide + A-R. The myocytes were preincubated first with HMR-1098 (30 µmol/l) for 10 min and then with HMR-1098 and diazoxide for 20 min before A-R.
Group 6: cyclosporin A + A-R.
The myocytes were preincubated with cyclosporin A (5 µmol/l), an
inhibitor of mitochondrial permeability transition, for 20 min before
A-R. This group was included to determine whether m has any effect on the opening of mitochondrial permeability transition pore.
Measurement of Cell Viability, Lactate Dehydrogenase, and ATP
Cell viability was calculated by dividing the number of trypan blue negative cells by the total number of cells examined and then multiplying by 100%. ATP was extracted by 6% trichloroacetic acid and analyzed at 340 nm in a Beckman spectrophotometer by using an ATP detection kit (Sigma). Lactate dehydrogenase (LDH) release from myocytes was measured by using a LDH detection kit (Sigma).Detection of Apoptosis and Distribution of Cytochrome c
To visualize apoptotic nuclei in cardiac myocytes in situ, the ApoTag in situ apoptosis detection kit (Oncor) was used. The cultured myocytes were fixed in 4% paraformaldehyde (pH 7.4) and subjected to TdT-mediated dUTP nick-end labeling (TUNEL) assay (36).The release and distribution of cytochrome c in intact myocytes were assayed as described by Xu et al. (36). Cultured myocytes on coverslips were fixed in 2% formaldehyde and blocked with 10% normal goat serum. Cells were stained with mouse monoclonal anti-cytochrome c as the primary antibody and fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G as the secondary antibody. For mitochondrial staining, unfixed cells were incubated with 500 µmol/l Mito Tracker Orange CMTMRos for 30 min at 37°C. After being washed and then fixed in 2% formaldehyde, the cells were observed with the use of a laser scanning confocal microscope (LSM 510, Zeiss).
Mitochondrial Morphology
The mitochondrial ultrastructure was assessed by transmission electron microscopy. Myocytes cultured on the coverslips were immersed in 2.5% buffered glutaraldehyde and rinsed in 0.1 mol/l sodium cacodylate buffer (pH 7.3). The cells were embedded in epon resin and cut into 600-nm-thick sections with a Sorvall MTB2 ultramicrotome. The sections were stained with uranyl acetate and lead citrate and examined with a Hitachi H-600 electron microscope at 75 kV.Mitochondrial Membrane Potential
The changes inm were monitored with the dye
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine
iodide (JC-1) (24). Cells were stained with JC-1 (5 µmol/l) at 37°C for 15 min and rinsed three times with Tyrode
solution. The observation was made by using a laser scanning confocal
microscope. JC-1 monomer (green) fluorescence was observed by
excitation with the 488-nm laser and examination of the emissions from
505 to 530 nm. JC-1 aggregate (red) fluorescence was observed by
excitation with the 543-nm laser and examination of the emissions over
560 nm.
One hundred or more areas were selected from each image and the average
intensity for each region was quantified (Metamorph, Universal Imaging;
West Chester, PA). The ratio of JC-1 monomer to aggregate intensity for
each region was calculated. An increase in this ratio was interpreted
as decrease of m, whereas a decrease in the ratio was
interpreted as gain in m (31).
Statistical Analysis
All data, except for them data, were
obtained in at least three independent experiments with replicates of
two or four for each condition. The m data were
obtained from 2-3 experiments, and 10-14 images were analyzed
in each group. Each image used for the m data
contained over 10 myocytes and 100 areas. All data were expressed as
means ± SE. Statistical significance between groups was
determined by Student's t-test. A value of
P < 0.05 was considered significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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MitoKATP Channel Activation Prevents A-R Injury
The protection by diazoxide on A-R-induced myocyte damage is shown in Figs. 2-4. Ninety percent of the control cells excluded trypan blue, whereas, in the A-R group, only 35% of the myocytes were viable (Fig. 2). LDH release (Fig. 3A) was significantly increased and ATP content (Fig. 3B) was significantly depleted after myocytes underwent A-R. Diazoxide-induced activation of the mitoKATP channel before A-R reduced cell death, decreased LDH release, and preserved ATP content. The protection provided by diazoxide was similar to that of cyclosporin A.
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To test whether diazoxide was activating KATP channels that were in the mitochondria or in the sarcolemma, the effect of diazoxide was examined in the presence of 5-HD, an inhibitor of mitoKATP channel, or HMR-1098, a sarcolemmal KATP channel inhibitor. The protective effect of diazoxide was decreased in the presence of 5-HD but not in the presence of HMR-1098.
MitoKATP Channel Activation Prevents Apoptosis
TUNEL assay was used to determine A-R-induced apoptosis. Less than 10% of the control myocytes had TUNEL-positive nuclei (Fig. 4). A-R significantly increased the number of TUNEL-positive nuclei. Diazoxide pretreatment reduced TUNEL-positive nuclei by 50%. The protective effect of diazoxide was similar to that of cyclosporin A.It has been indicated that cytochrome c release from
mitochondria is followed by apoptosis. Cytochrome c
immunostaining (Fig. 5B)
coincided with the distribution of mitochondria in control myocytes
(Fig. 5A). After A-R, there was diffuse cytochrome
c immunostaining in some cells, which suggested the release
of cytochrome c into the cytosol (Fig. 5E).
Pretreatment of myocytes with diazoxide significantly blocked the
release of cytochrome c (Fig. 5H). 5-HD reversed
the action of diazoxide and HMR-1098 did not significantly inhibit the
protection by diazoxide.
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MitoKATP Channel Activation Protects Mitochondrial Morphology
When examined with the electron microscope, mitochondria were observed in rows between myofibrils or were scattered loosely throughout the cytoplasm (Fig. 6A). After A-R, mitochondria became swollen and cristae were disrupted and contained electron dense deposits (Fig. 6B). When myocytes were treated with diazoxide before A-R, the mitochondrial structure was markedly preserved (Fig. 6C).
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MitoKATP Channel Activation Restores
m
m. Green fluorescent mitochondria were localized near
the nucleus, whereas red fluorescent mitochondria were confined to the
cell periphery (Fig. 7C).
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Immediately after shorter A-R (i.e., 2-h anoxia and 2-h reoxygenation),
the first response of mitochondria was hyperpolarization (unpublished
observations). After A-R with a longer anoxic period (3 h), myocytes
showed marked changes in m. Many myocytes displayed a
loss or collapse of m, as evident from the
disappearance of red or both red and green fluorescence in several
cells (Fig. 7F) or mitochondria were condensed into an
extremely packed mass (Fig. 7E). These changes were
accompanied by apoptosis-associated morphological changes,
such as nuclear chromatin condensation, the reduction of cell volume
(Fig. 6), and the release of cytochrome c (Fig. 5). Many
other myocytes displayed elongated mitochondria in the cell periphery
and highly polarized mitochondria in the cell center (Fig. 7,
D and F). Pretreatment of the myocytes with diazoxide protected mitochondria from the loss of m
and from hyperpolarization (Fig. 7, G-I).
Whereas many cells displayed a loss or collapse of m,
many other cells (perhaps less damaged) displayed an increase in
m in response to A-R. The ratio of JC-1 monomer
(green) to aggregate (red) fluorescence was used to quantify
m in these less damaged cells. Myocytes with
extremely packed mitochondria and myocytes lacking red fluorescence
were considered severely damaged and were excluded from analysis. With
A-R, the JC-1 ratio was reduced (m increased)
compared with the control myocytes (Fig.
8). The JC-1 ratio in
diazoxide-pretreated cells was maintained at a level that was similar
to that of control and of cyclosporin A-pretreated cells (Fig. 8). The
diazoxide-induced maintenance of m was reduced by the
mitoKATP inhibitor 5-HD but not by the sarcolemmal
KATP channel inhibitor HMR-1098.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Mitochondria are the critical organelle for myocyte cell survival.
A compromise of mitochondrial function during A-R may lead to cell
demise. The results of this study strongly support the notion that
m is severely disrupted during A-R and is accompanied by leakage of cytochrome c, apoptosis, destruction
of cristae, and accumulation of Ca2+ in mitochondria.
Our data indicate that reoxygenation after anoxia produced a variable
but often profound loss of m. A loss in
m is accompanied by cytochrome c release
from the mitochondria and leads to induction of apoptosis in
different cell types (1, 28). Loss of m is correlated with the release of cytochrome c from
mitochondria as determined by immunostaining. Therefore, it is highly
likely that loss or dissipation of m mediated the
release of cytochrome c, which activated apoptosis.
Bialik et al. (3) also presented similar evidence of a
mitochondrial apoptotic pathway during ischemia. Postanoxic
reoxygenation also caused a significant elevation of intramitochondrial
Ca2+, resulting in loss of m (7,
10).
In our study, many cells indicated high polarized mitochondria after A-R. This hyperpolarization may result from lower oxygen availability or be generated by reverse ATP synthase activity supported by glycolytic ATP (4, 8). However, what seems evident is that mitochondrial high polarization is not synonymous with enhanced mitochondrial activity (9). Indeed, an inverse relationship may be assumed between the average level of mitochondrial polarization and ATP synthesis (33), at least in intact cells, where complex homeostatic mechanisms are established between mitochondria and other cytoplasmic compartments. High polarization of mitochondria may be the force to drive Ca2+ inside mitochondria, which will induce Ca2+ overload in mitochondria and trigger apoptosis (21). Moreover, it has been reported (25) that first the mitochondrial membrane potential increases before cytochrome c release that occurs after a loss in potential and mitochondrial swelling.
The present study demonstrated that diazoxide stabilized
m by attenuating the loss of m and
high polarization observed during A-R. Holmuhamedov et al.
(13) reported that mitoKATP channel openers
caused concentration-dependent mitochondrial membrane depolarization in
normal cultured myocytes. At a low concentration (100 µmol/l) of the
KATP channel opener pinacidil, m of
myocytes was decreased by 10 mV (13). However, a high
concentration (1 mmol/l) of KATP channel opener decreased
m over 150 mV. Similarly, diazoxide decreased
m in mouse intact perfused pancreatic B cells and
isolated liver mitochondria, accelerating the release of
Ca2+ stored in the mitochondria (12).
Activation of the mitoKATP channel with diazoxide (100 µmol/l) results in K+ influx, expansion of mitochondrial
matrix volume, and a reduction of the inner m
(13). Depolarization of the m reduced
the driving force for Ca2+ influx (6, 14),
thus attenuating mitochondrial Ca2+ overload and myocyte
injury during A-R (30). Our study indicates that the
stabilization of m by activation of
mitoKATP channel was accompanied by remarkable recovery of
ATP and absence of Ca2+ accumulation in mitochondria.
The precise mechanism of diazoxide on m remains
unknown. There are multiple electron transport systems in mitochondria
that are disrupted by A-R, and opening mitoKATP channels
will facilitate homeostasis of electron transport system. McPherson and
Yao (23) recently showed that 
-opioid receptor
stimulation opens mitoKATP channels, resulting in a small
increase of reactive oxygen species. These reactive oxygen species are
important components of mitochondrial transmembrane potential and
participate in signaling cascade leading to cardioprotection. In
cardiac myocytes, m may be regulated via activation
of KATP channel. Diazoxide prevented high polarization of
the mitochondrial membrane and the collapse of m,
thus inhibiting the Ca2+ accumulation by mitochondria. This
was further substantiated by use of a selective KATP
channel blocker, 5-HD, which prevented the depolarizing action of a
mitoKATP channel opener. In another study
(13), it was reported that KATP channel
activation decreased ATP synthesis and released mitochondrial proteins.
Both of these parameters are associated with cell death. However,
overwhelming evidence (19, 20, 28) suggests that
apoptosis is mediated by the cytochrome c released
from the mitochondria. It is not clear how diazoxide-induced cytochrome
c release can protect against ischemic injury, as
reported by Holmuhamedov et al. (13). Our previous studies
(36) have indicated that the concentration of cytochrome
c in mitochondria displayed a negative linear correlation with the percentage of apoptosis in the myocytes. In the
present study, diazoxide prevented cytochrome c release from
myocytes subjected to A-R, suggesting a role of mitoKATP
channel in cardiac protection. This confirms our previous findings
(30) that diazoxide inhibits both apoptosis and
necrosis in late preconditioning.
The effect of diazoxide on m was comparable with that
observed after cyclosporin A treatment. The reduced m
increases the likelihood of opening of mitochondrial permeability
transition pore which is prevented by cyclosporin A. Outward pumping of
protons at mitochondrial respiratory complexes I, III, and IV generates m across the inner mitochondrial membrane
(27) and decreased m facilitates opening
of permeability transition pore (26). As suggested by our
data, there appears to be commonality of m between
opening of mitochondrial KATP channel and cyclosporin A-sensitive permeability transition pore.
In summary, the mitoKATP channel activation prevented the
myocyte damage caused by A-R. Opening of mitoKATP channel
stabilized the m in anoxic myocytes, resulted in
marked augmentation of cell ATP, and inhibited the loss of cytochrome
c from mitochondria leading to attenuation of
apoptosis. Thus maintenance of mitochondrial function and
structural integrity by diazoxide suggests a potential therapeutic
application of mitoKATP channel agonists in preventing ischemic injury.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Nancy K. Kleene, Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati, for assistance with confocal microscopy and helpful discussions.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-23597 and HL-55678.
Address for reprint requests and other correspondence: M. Ashraf, Dept. of Pathology and Laboratory Medicine, Univ. of Cincinnati, 231 Bethesda Ave., Cincinnati, OH 45267-0529 (E-mail: Muhammad.Ashraf{at}UC.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 April 2001; accepted in final form 17 May 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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P < 0.05 vs. DZ + A-R.
