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Introduction
Schisandrin B (Sch B, Figure 1) is the most abundant, active dibenzocyclooctadiene derivative isolated from the fruit of
Schisandra chinensis (Turcz) Baill, a traditional Chinese herb clinically used for the treatment of viral and chemical
hepatitis[1]. Previous studies in our laboratory have de-monstrated the ability of Sch B to protect against myocardial
ischemia-reperfusion (I_R) injury[2]. The cardioprotection afforded by Sch B pretreatment were associated with the enhancement in
tissue glutathione antioxidant status, particularly in the
mitochondrion[2_4]. Recent studies also showed that Sch B protected
against myocardial I_R injury partly by inducing heat shock protein (Hsp)25 and Hsp70
expression in rats[5]. However, it is still
unclear whether Sch B treatment produces any effect on the sensitivity of the mitochondria to permeability transition (PT).
Growing evidence has accumulated suggesting the involvement of PT of the mitochondrial inner membrane in the
pathogenesis of oxidant injury in various tissues, including myocardial I_R
injury[6,7]. One critical event of mitochondrial PT is the
permeability of the inner membrane to small ions and solutes with
Mr <1500 daltons, with the consequent, large amplitude
swelling of the mitochondria[8]. The opening of mitochondrial PT pores plays an important role in regulating necrotic and
apoptotic cell death[9]. While the loss of ion homeostasis
resulting from ATP depletion following PT can lead to
necrosis[10]. PT also causes the leakage of cytochrome c from the
mitochondria to the cytosol[11]. The released cytochrome c
can trigger a cascade of events that eventually lead to
apoptosis[11,12].
Increased Ca2+, reactive oxidant species (ROS), and
inorganic phosphate, as well as decreased membrane potential,
have been shown to activate mitochondrial PT, whereas
Ca2+ chelator, antioxidants, such as glutathione and ubiquinone
analogs, ATP/ADP and other translocase ligands, such as
cyclosporin A (CsA), inhibit mitochondrial
PT[13,14]. In order to further elucidate the molecular mechanism underlying the
cardioprotective action of Sch B, we endeavoured to
investigate the effect of Sch B treatment on the sensitivity of the
mitochondria to Ca2+-stimulated PT in rat hearts under normal and
I_R conditions. Changes in mitochondrial
Ca2+ content, ROS production, and cytochrome c release were also examined in
relation to alterations in the sensitivity to mitochondrial PT.
Materials and methods
Chemicals and herbal material CsA was purchased from
Sigma Chemical Co (St Louis, MO, USA). All other
chemicals were of analytical grade. The dried fruits
of Schisandra chinensis were imported from mainland China. They was
authenticated and supplied by a commercial dealer (Lee
Hoong Kee Limited, Hongkong, China) in Hong Kong. Sch
B was purified from the petroleum ether extract of
Schisandra fruits, with the purity being higher than 95% as determined
by HPLC analysis[15].
Animal care Sprague-Dawley rats (8_10 weeks old,
weighing 250_300 g) were maintained under a 12 h dark/light
cycle at about 22 oC and allowed food and
water ad libitum in the Animal Care Facilities at the Hong Kong University of
Science and Technology (HKUST, Hong Kong, China). All
experimental protocols were approved by the University
Committee on Research Practice at HKUST.
Drug treatment The animals were randomly divided into
groups of 5 animals. In the pretreatment groups, the rats
were treated intragastrically with Sch B
(dissolved/suspended in olive oil) at a single dose of 1.0 or 2.0 mmol/kg.
Untreated controls received the vehicle only. The hearts
were isolated from the rats and subjected to I_R experiment
48 h following the administration of Sch B. Previous studies
indicated that the optimal single dose of Sch B for protecting
against myocardial I_R injury ranged from 1 to 2 mmol/kg,
and the maximum protective effect was observed at 48 h
post-dosing with Sch B[3].
Isolated-perfused rat hearts The hearts were excised
quickly from the phenobarbital-anesthetized rats and
immediately immersed in ice-cold and heparinized (50 unit/mL)
saline. The aorta was cannulated and then transferred to a
warm, moist chamber of the perfusion apparatus. The hearts
was retrogradely perfused, with the coronary flow rate
ranging from 8 to 12 min, according to the Langendorff method as
described[16].
Myocardial I_R After an initial 30 min of perfusion for
equilibration, the isolated hearts were subjected to a 40 min
period of "no-flow" global ischemia followed by 20 min
reperfusion. Coronary effluent was collected in 1 min
fractions at increasing time intervals during the course of
equilibration and reperfusion. The fractions were immediately
placed on ice until assayed for lactate dehydrogenase (LDH)
activity. The extent of LDH leakage during the reperfusion
period, an indirect index of myocardial injury, was estimated
by computing the area under the curve (AUC) of the graph
plotting the percentage of LDH activity (with respect to the
mean pre-ischemic value measured during the equilibration
period) against the reperfusion time (1_20 min) as described[4], and the value was expressed as arbitrary units (AU).
Non-I_R hearts were perfused for 90 min. After the non-I_R
or I_R procedure, ventricular tissue samples were obtained
and subjected to biochemical analysis.
Preparation of mitochondrial fractions and cytosolic
fractions Myocardial ventricular tissue samples were
rinsed with ice-cold isotonic buffer [210 mmol/L mannitol,
70 mmol/L sucrose, 5 mmol/L HEPES, 1 mmol/L EGTA (pH 7.4) 0.2 mg/mL soybean trypsin inhibitor, 0.2 mg/mL
bacitracin, and 0.16 mg/mL benzamidine]. Myocardial
tissue homogenates were prepared by homogenizing 0.8 g
minced tissues in 8 mL ice-cold isotonic buffer. The
mitochondrial fractions were prepared by differential
centrifugation as described[17]. The homogenates were
centrifuged at 600×g for 20 min. After collecting the supernatants,
the pellets were resuspended with the same volume of
ice-cold homogenizing buffer (without various protease
inhibitors) and recentrifuged at 600×g again. The procedure
was repeated twice. The pooled supernatants (a total of 4
volumes) were centrifuged at 8000×g for 30 min, and the
mitochondrial pellets were collected. The supernatant was saved
for the preparation of the cytosolic fraction. The
mitochondrial pellets were then washed with the same volume of
ice-cold sucrose buffer containing 210 mmol/L mannitol, 70
mmol/L sucrose, and 5 mmol/L HEPES_KOH (pH 7.4) and the
mixtures were centrifuged at 8000×g for 30 min. The washing
procedure was repeated. The mitochondrial pellets were
resuspended in 0.5_1.0 mL ice-cold sucrose buffer and
constituted the mitochondrial fractions. The mitochondrial
fractions, prepared from various rat tissues by a similar
protocol in our laboratory, were found to carry out oxidative
phosphorylation in a normal manner[18]. The cytosolic
fraction was prepared by centrifuging the above supernatant at
100 000×g for 60 min at 4 oC.
Mitochondrial ROS generation The extent of
mitochondrial ROS generation in vitro was estimated as an indirect
measure of mitochondrial antioxidant capacity and structural
integrity. An aliquot (50 µL) of mitochondrial fraction (50 µg
protein/mL) and 60 µL 2',7'-dichlorofluorescin diacetate
(DCFDA; Fluka, Switzerland) solution (5 µmol/L in
incubation buffer containing 0.1% DMSO) were added into the
wells of a black microtiter plate. The mixture was
incubated at 37 oC for 10 min under dark condition in a
Victor2 Multi-Label Counter (Perkin_Elmer, Wellesley, MA, USA).
After the incubation, 50 µL incubation buffer [0.1 mmol/L
EGTA, 5 mmol/L KH2PO4, 3 mmol/L
MgCl2, 145 mmol/L KCl, 30 mmol/L HEPES (pH 7.4)] and 50 µL substrate solution (20
mmol/L pyruvate and 10 mmol/L malate) were added.
Fluorescence intensity (excitation at 485 nm and emission at 535
nm) of the reaction mixture was monitored every 5 min for 30
min. The mitochondrial ROS generation was reflected by the
fluorescence intensity of the sample after subtracting the
value of a blank sample containing incubation buffer,
substrate solution, and DCFDA. The extent of ROS generation
over the 30 min period of incubation was estimated by
computing the AUC of the graph plotting fluorescent intensity
against time (0_30 min) and expressed as an AU.
Mitochondrial PT The measurement of mitochondrial
PT was performed according to a procedure modified from
Kowaltowski et al[19]. An aliquot (1.6 mL) of the
mitochondrial sample (0.5 mg protein/mL) was prepared by mixing the
mitochondrial fraction with incubation buffer containing 125
mmol/L sucrose, 65 mmol/L KCl, 10 mmol/L HEPES (pH 7.2),
5 mmol/L freshly prepared succinate, and 5 µmol/L freshly
prepared rotenone. Aliquots (200 µL) of the mitochondrial
sample were mixed with 10 µL of CsA (5 μmol/L) in 0.5%
(w/w) ethanol final concentration or incubation buffer. The
mixtures were incubated at 30 oC for 5 min. An aliquot (10 µL)
of calcium chloride (Ca2+) solution (1 µmol/L final
concentration) was then added, and the mixtures were
incubated at 30 oC for 5 min. Aliquots (180 µL) of the mixtures
were added into a 96-well microtiter plate, and the initial
absorbance of the mixtures at 520 nm was monitored for 5 min
at 30 oC. The swelling reaction was then started by adding
20 µL potassium phosphate (0.5 mmol/L, pH 7.2), and the
absorbance at 520 nm of the reaction mixtures was read
every 2 min for 30 min at 30 oC using the Victor
V2 Multi-Label Counter. The extent of mitochondrial swelling was estimated
by computing the AUC of the declining graph plotting the
percentage of the initial absorbance (100% as baseline)
against time (min) to obtain AUC1. The extent of
mitochondrial PT (ΔAUC1) was estimated by subtracting the
AUC1 with CsA from the AUC1 without CsA.
Ca2+-induced mitochondrial PT was expressed in a ratio of
ΔAUC1 induced by both Ca2+ and
PO43_ to that induced by
PO43_ only.
Mitochondrial Ca2+ content The
Ca2+ content was measured using a
Ca2+-sensitive fluorescence probe Fluo-5N AM
ester (Molecular Probe, OR, USA) by the
Victor2 V Multi-Label Counter, as described in
Menze et al[20]. The
Ca2+ dissociation constant
(Kd) was determined by using a
Ca2+ calibration kit at a concentration range of 1_1000 µmol/L,
with the Kd value estimated at being ~98 µmol/L, which is in
agreement with the data provided by the manufacturer.
An aliquot (25 µL) of the mitochondrial fraction (0.5
mg/mL final concentration) was mixed with 25 µL incubation buffer (100
mmol/L KCl and 30 mmol/L MOPS, pH 7.2) in a 96-well black
microtiter plate. The mixture was incubated at 25
oC for 15 min and then added with 25 µL digitonin (50 µg/mL) and 25
µL Fluo-5N AM ester (1 µmol/L in 0.005% Pluronic F-127).
Presumably, by permeating the membrane, digitonin can
facilitate the entry of the fluorescence dye into the
mitochon-dria. Experimental data indicated that the fluorescence
intensity of Fluo-5N measurable in rat heart mitochondrial
preparations was increased by 20-fold in the presence of
digitonin. The reaction mixture was incubated at 25
oC for 30 min, and the fluorescence reading was measured at the
excitation wavelength of 488 nm and emission at 532 nm. The
mitochondrial Ca2+ content was estimated from the standard
calibration curve and expressed in μmol/mg protein.
Biochemical analysis The LDH activity of coronary
effluent was measured as described[4]. The cytosolic
cytochrome c level, as an indirect measure of mitochondrial
cytochrome release, was estimated by Western blot analysis
using specific antibodies of cytochrome c (clone 7H8.2C12,
BD PharMingen, San Diego, CA, USA) following SDS_PAGE
analysis of the cytosolic fractions using a separating gel of
15% acrylamide. The extent of mitochondrial contamination
in the cytosolic fractions was determined using specific
antibodies against complex IV. The immunoblots were
visualized by enhanced chemiluminescence reaction (Amersham
ECL+, Piscataway, NJ, USA) and analyzed by
densitometry (Bio-Rad). The amounts (AU) of cytochrome c were
normalized with reference to actin levels (AU) in the sample.
The protein concentrations of the mitochondrial fractions
were determined using a Bio-Rad protein assay kit (Hercules,
CA, USA).
Statistical analysis Data were analyzed by one-way
ANOVA. Post-hoc tests for pair-wise multiple comparisons
were done with Least Significant Difference, using SPSS
statistical software (SPSS, Chicago, USA).
P-values <0.05 were regarded as statistically significant.
Results
Ca2+, when added at a final concentration of 1 µmol/L in
the presence of PO43_, enhanced the time-dependent decrease
in absorbance at 520 nm, an indirect measure of
mitochondrial swelling, in a suspension of heart mitochondria (Figure
2). The Ca2+-stimulated mitochondrial swelling was largely
inhibited by CsA (5 µmol/L). While mitochondria from
non-I_R and I_R hearts exhibited a comparable and small extent
of swelling in the absence of Ca2+, they showed a larger
extent of swelling in the presence of
Ca2+, with the extent of
Ca2+-induced PT of I_R mitochondria being larger than that
of the non-I_R control. The extent of CsA-inhibitable
mitochondrial swelling induced by Ca2+ was computed and
served as an indirect measure of mitochondrial PT, as
described in Materials and methods.
Pretreatment with Sch B (1_2 mmol/kg, po) protected
against myocardial I_R injury in rats, as evidenced by the
significant decrease in the extent of LDH leakage (23%_38%),
when compared with the unpretreated control (Figure 3A).
While Sch B treatment at the same dosage suppressed
Ca2+-stimulated PT in heart mitochondria (7%_15%), when
compared with the untreated control, I_R caused a slight, but
significant increase in the sensitivity of heart mitochondria
to Ca2+-stimulated PT (by 18%) in the rats (Figure 3B). The
inhibitory effect of Sch B treatment on mitochondrial PT was
also evident after the I_R challenge. The pretreatment with
Sch B significantly reduced the extent of mitochondria PT
(by 9%_18%) in I_R hearts, when compared with the unpretreated I_R control (Figure 3B).
I_R caused significant increases in the mitochondrial
Ca2+ level (21%), as well as the extent of mitochondrial ROS
production (15%) and cytochrome c release (67%) the in rat
hearts (Figure 4A_4C). While Sch B treatment
dose-dependently decreased the mitochondrial
Ca2+ level (by 35%_55%) in non-I_R rat hearts (Figure 4A), the extent of mitochondrial
ROS production and cytochrome c release remained
relatively unchanged (Figure 4B,4C). Sch B pretreatment
dose-dependently suppressed the I_R-induced increases in the
mitochondrial Ca2+ level, ROS production, and cytochrome c
release to varying degrees (71%_85%, 23%_30%, and
19%_24%, respectively) when compared with the respective Sch
B unpretreated control (Figure 4A_4C).
Discussion
Mitochondria not only generate ATP through oxidative
phosphorylation, but are also an important source of ROS in
mammalian cells under both
physiological[21] and pathological conditions, such as myocardial I_R
injury[22]. As far as the maintenance of cellular structural and functional
integrity is concerned, the mitochondria serve as coordinators for
cell survival and death[23]. In this regard, the opening of the
mitochondrial transition pore is critically involved in the
development of cellular dysfunction and cell
death[24]. In the present study, the I_R-induced myocardial injury was
associated with the increase in the sensitivity of the
mitochondria to Ca2+-induced PT, as assessed
by in vitro measurement of mitochondrial swelling. Core components
of mitochondrial PT pore putatively consist of a
voltage-dependent anion channel, an adenine nucleotide translocase
(ANT), and cyclophilin D that displays peptidyl-prolyl
cis-trans isomerase (PPIase)
activity[7]. An excess amount of
Ca2+ would compete over ATP and activate PPIase, resulting
in a conformation change that converts the ANT complex
into a non-specific pore[25]. The opening of mitochondrial
PT pores, either in in vivo or in
vitro conditions, is triggered by high mitochondrial
Ca2+ content and other stimuli,
including oxidants and the depletion of adenine
nucleotides[26]. Consistent with this, mitochondria isolated from I_R rat
hearts were found to have increased
Ca2+ content as well as increased extent of ROS production. The disruption of
structural integrity of mitochondria, as implicated by increases in
the sensitivity to mitochondria PT and the extent of
mitochondrial ROS production, from I_R myocardial tissues was
evidenced by the increase in cytochrome c release
in vivo. The cardioprotection afforded by Sch B pretreatment against
I_R injury was associated with the improvement in
mitochondrial integrity, as indicated by decreases in the
sensitivity to Ca2+-induced PT, as well as the extent of ROS
production and cytochrome c release. This is also corroborated
by our recent findings that showed the preservation of
mitochondrial ATP generation capacity by Sch B pretreatment in
I_R rat hearts[3]. The leakage of cytochrome c in non-I_R
hearts, as evidenced by the presence of cytosolic cytochrome
c, is likely caused by the artifact arising from tissue
homoge-nization, as was the case for Kim et
al[27].
Cytosolic Ca2+ content increases during myocardial
I_R[28], leading to the accumulation of
Ca2+ in the mitochondria via the uptake by the inner membrane
Ca2+ uniporter[29,30]. The mitochondrial
Ca2+ overload not only generates energy-consuming futile cycles that divert the use of the inner
membrane proton gradient to cation transport rather than ATP
production[30], but also predisposes the mitochondria to
PT[31]. The mitochondrial PT further jeopardizes the cellular energy
status, and the consequent loss of ion homeostasis can lead
to necrotic cell death. The release of cytochrome c from the
mitochondrial inner membrane, which is believed to be an
event secondary to the onset of mitochondrial
PT[32], is a key step leading to
apoptosis[11]. While under the present
experimental conditions the relative contribution of necrotic
and apoptotic cell death to I_R-induced tissue injury remains
to be determined, the finding of decreased sensitivity of
mitochondria isolated from Sch B-pretreated hearts to
Ca2+-stimulated PT suggests that the increase in mitochondrial
resistance to Ca2+-stimulated PT may play an important role
in protecting against myocardial I_R injury. The
observation that Sch B treatment decreased the mitochondrial
Ca2+ level in both non-I_R and I_R hearts may be caused by the
inhibition of the inner membrane Ca2+ uniporter and hence
the Ca2+ uptake by Sch B. In this regard, Sch B was found to
inhibit the P-glycoprotein, an ATP-dependent drug
transporter in cancer cells[33].
Glutathione plays an important role in numerous cellular
functions, including the regulation of
Ca2+ homeostasis and detoxification of
ROS[34,35]. Previous studies have
demonstrated the ability of Sch B to enhance mitochondrial
gluta-thione redox status and protect against I_R-induced
myocardial injury[3]. The decrease in mitochondrial sensitivity to
Ca2+-stimulated PT by Sch B, as observed in the present
study, may be related to the enhancement of mitochondrial
glutathione redox status. In this regard, 1 of the 2
voltage-sensitive sites of mitochondrial PT pore was found to be
gated by a critical dithiol that was sensitive to glutathione
redox status, and the oxidation of GSH could open the
pore[36,37]. Furthermore, the modification of a specific thiol group on
the ANT, either by oxidative stress or thiol reagents, also
decreased adenine nucleotide binding and activated the PT
pore[38]. Increased oxidative stress enhanced cyclophilin
binding and hence increased the sensitivity of
mitochondrial PT[38]. Consistently, the cardioprotection against I_R
injury and the increase in the resistance of mitochondria to
Ca2+-induced PT afforded by Sch B pretreatment were
abolished by GSH depletion (unpublished data). The
observation that a smaller degree of inhibition of
Ca2+-induced mitochondrial PT than that of myocardial I_R injury afforded by
Sch B treatment supports the notion that the decrease in the
sensitivity of mitochondria to PT is an effect secondary to
the enhancement of mitochondrial glutathione redox status.
The direct effect of increasing the mitochondrial GSH level
and/or the indirect effect on removing mitochondrial-derived
ROS produced by Sch B treatment may probably increase
the threshold for mitochondrial PT pore opening in the
presence of Ca2+. It is unlikely that Sch B can produce a direct
effect on mitochondrial PT pore opening because Sch B,
when being supplemented in the perfusate, could not
protect against I_R injury in isolated rat
hearts[4].
In conclusion, I_R caused an increase in the sensitivity
of myocardial mitochondria to Ca2+-stimulated
PT in vitro. The cardioprotection afforded by Sch B pretreatment against
I_R injury was paralleled by the decrease in the sensitivity
of the myocardial mitochondria to Ca2+-stimulated PT,
particularly under I_R conditions. The results suggest that the
cardioprotection afforded by Sch B pretreatment against
I_R injury may at least in part be attributed to the increase in
the resistance of the myocardial mitochondria to
Ca2+-stimulated PT.
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