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INTRODUCTION |
Heat shock proteins
(HSPs)1 are a family of
chaperone proteins induced by hyperthermia, oxidative stress,
ischemia/reperfusion, hypoxia, energy depletion, viral infection, UV
radiation, proinflammatory cytokines like tumor necrosis factor-
,
and other stress inducers (for review see Ref. 1). HSPs provide
tolerance against both thermal and oxidative stress (1, 2) and inhibit
cell death caused by H2O2 (3, 4), ATP depletion
(5), Fas ligand (6), tumor necrosis factor-
(7), transforming growth
factor-
(8), endotoxin lipopolysaccharide (9), ceramide (10), and
ischemia-reperfusion injury (11, 12). In L929 cell lines, overexpression of human Hsp27, Drosophila Hsp27, or human
B-crystalline decreases endogenous reactive oxygen species (ROS)
production and abolishes the burst of intracellular ROS induced by
tumor necrosis factor-
(7). In vitro, Hsp70 and Hsp27
inhibit cytochrome c-mediated procaspase 9 processing either
by blocking cytochrome c release from mitochondria or by
binding the released cytochrome c in the cytosol, thus
blocking the apoptotic pathway (13, 14).
The protection by HSPs against such varied stimuli suggests a common
pathway of protection. A preferential target of HSPs protection may be
mitochondria. Overexpression of the mitochondrial HSPs, Hsp60 and
Hsp10, protects cardiac myocytes from ischemia/reperfusion injury
through maintaining mitochondrial integrity and function (15). Reduced
expression of Hsp60 by an antisense oligonucleotide precipitates
apoptosis, which is accompanied by cytochrome c release from
mitochondria and caspase activation (16). Heat shock treatment also
prevents decreases of state 3 respiration in isolated myocardial mitochondria and mitochondrial membrane potential in the U937 human
premonocytic cell line induced by H2O2 (4,
15).
Opening of mitochondrial permeability transition (MPT) pores plays an
important role in regulating apoptotic and necrotic cell death (17).
Increased Ca2+, ROS, ADP, and atractyloside activate the
MPT, whereas cyclosporin A (CsA), bongkrekic acid, Mg2+,
Ca2+ chelation, low pH, and ubiquinone analogues such as
ubiquinone 0 and decylubiquinone inhibit the MPT (18, 19). Recently, we
presented data that there are two open conductance modes for MPT pores:
a regulated mode that is activated by Ca2+ and inhibited by
CsA, and an unregulated open mode that does not require
Ca2+ and is not inhibited by CsA (20-24). In general, low
dose MPT induction opens regulated pores, whereas high dose
induction opens unregulated pores (20).
Despite evidence that HSPs protect mitochondria against injury, no
studies have investigated whether the MPT is involved in HSPs
protection. Here we investigated the consequence of heat shock to rats
on the MPT in isolated rat liver mitochondria. We show that heat shock
treatment inhibits the induction of regulated MPT pore opening and
increases the threshold of unregulated MPT pore opening, effects that
correlate with increased expression of mitochondrial Hsp25. The results
suggest that heat shock-inducible factors regulate against the onset of
the MPT, which may explain the protection by HSPs against cell injury.
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EXPERIMENTAL PROCEDURES |
Tetramethylrhodamine methyl ester (TMRM), Fluo-5N, and
2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA)
were obtained from Molecular Probes (Eugene, OR); antibodies to
inducible Hsp70 (Hsp72), Hsp10, Hsp25, Hsp60, and mitochondrial Hsp70
(Grp75) were from StressGen Biotechnologies (Victoria, British
Columbia, Canada); antibody to Hsp75 (tumor necrosis factor
receptor-associated protein 1: TRAP-1) was from Lab Vision Corp.
(Fremont, CA); antibody to voltage-dependent anion channel
(VDAC) was from Calbiochem; and custom-made anti-cyclophilin D (CypD)
antibody was from Bethyl Laboratory (Montgomery, TX). Other reagent
grade chemicals were obtained from Sigma.
Adult male Sprague-Dawley rats (250-300 g) that were fasted overnight
were used in all the experiments. For heat shock treatment, rats were
anesthetized (50 mg of pentobarbital/kg body weight) and immersed in a
42 °C water bath in plastic bags. Rectal temperature was monitored
using a digital thermometer. After rectal temperature reached 42 °C
after about 30 min, the rats were maintained at 42 °C for 15 min
before removing them from the water bath. Sham-treated rats were
subjected to anesthesia only. An additional control group received no
treatment. In preliminary experiments, Hsp72 expression in liver
homogenates was assessed by Western blotting from 0 to 40 h after
heat shock. These experiments showed that peak expression of Hsp72
occurred at 20 h (data not shown). Accordingly, subsequent
experiments were performed 20 h after heat shock and sham treatment.
Rat liver mitochondria were isolated by differential centrifugation and
resuspended at 50 mg of protein/ml in 200 mM sucrose and 2 mM HEPES, pH 7.4 buffer, as described previously (25). Mitochondria were either used immediately or aliquoted, frozen in
liquid nitrogen, and stored at
80 °C. In addition, supernatants of
the first 10,000 × g spin were centrifuged at
100,000 × g for 30 min at 4 °C. The high speed
supernatant representing the cytosolic fraction was frozen in liquid
nitrogen and saved for total glutathione measurement and Western
blot analysis. Protein concentrations in mitochondria and cytosol were
determined by a Biuret procedure using bovine serum albumin as standard
(26).
Mitochondrial oxygen consumption using succinate as a substrate was
assessed using a Clark-type oxygen electrode (27). Mitochondrial swelling was monitored at 25 °C by absorbance at 540 nm with a ThermoMax 96-well plate reader (Molecular Devices, Sunnyvale, CA) in
incubation buffer containing 200 mM sucrose, 20 µM EGTA, 5 mM succinate, 2 µM
rotenone, 1 µg/ml oligomycin, 20 mM Tris, 20 mM HEPES, and 1 mM
KH2PO4, pH 7.2, and the MPT was induced by
CaCl2, HgCl2, and mastoparan, as described
previously (20). Mitochondrial membrane potential and Ca2+
uptake/release were monitored using 1 µM TMRM and 1 µM Fluo-5N, respectively, as described previously (27).
ROS formation was monitored fluorometrically after ester loading
mitochondria with 10 µM H2DCFDA from the rate
of increase of the green fluorescence of DCF, the oxidized product of
H2DCF. Total glutathione in frozen mitochondria and
cytosolic fractions was quantified using the Bioxytech GSH-420 kit
(Oxis Research, Portland, OR) according to the manufacturer's instructions.
To measure the heat shock proteins Hsp10, Hsp25 (rodent homologue of
human Hsp27), Hsp60, Hsp70, Hsp75, and the putative MPT pore complex
proteins CypD and VDAC in cytosolic and mitochondrial fractions,
proteins were resolved by 8-15% SDS-PAGE and transferred to
polyvinylidene difluoride membranes. Western blotting was carried out
and developed using the ECL Plus kit (Amersham Biosciences) according
to the manufacturer's instructions. Signals were imaged using a
Molecular Dynamics Storm Image System (Eugene, OR).
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RESULTS |
Heat Shock Inhibits the MPT Induced by Low and High Levels of MPT
Inducers--
To determine the possible role of heat-inducible factors
in MPT regulation, liver mitochondria isolated from control,
sham-control, and heat shock-treated rats were incubated with low and
high doses of MPT inducers. MPT was monitored by swelling detected by
absorbance at 540 nm. In control mitochondria, a low dose of
HgCl2 (5 µM) induced rapid mitochondrial
swelling in the presence of 50 µM CaCl2 (Fig.
1A, trace a).
Swelling was half-maximum after about 5 min of addition of
HgCl2 plus CaCl2 and was maximum after about 8 min. MPT induction in mitochondria isolated from control and sham
control rats was not different, so only the results from the control
group are shown. By contrast, in mitochondria isolated from rats
subjected to heat shock, the Ca2+-dependent MPT
produced by low dose HgCl2 induction was substantially delayed. In the heat shock group, half-maximum swelling was reached more than 15 min later than in the control group (Fig.
1A, trace b).

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Fig. 1.
Heat shock treatment delays onset of the
regulated MPT and increases the threshold of unregulated MPT induced by
HgCl2. Mitochondria (0.5 mg of protein/ml) were added
to incubation buffer, and the onset of the MPT was monitored by
absorbance, as described under "Experimental Procedures." After a
2-3-min preincubation, 5 µM HgCl2 plus 50 µM CaCl2 (A), 20 µM
HgCl2 (B), and 40 µM
HgCl2 (C) were added. Traces a and
b are control mitochondria and heat shock-treated
mitochondria, respectively. The data shown are representative of four
independent experiments.
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We also induced the unregulated MPT by exposure of mitochondria to
higher doses of HgCl2 in the absence of added
Ca2+. In control mitochondria, large amplitude
mitochondrial swelling began immediately after the addition of 20 µM HgCl2 and reached half-maximum within 5 min (Fig. 1B, trace a). Again, there was no
difference between the control and sham control groups (data not
shown). After heat shock, half-maximal swelling was delayed about 15 min (Fig. 1B, trace b). When the concentration of
HgCl2 was increased to 25 and 30 µM, heat
shock treatment still delayed onset of the MPT (data not shown). By
contrast, after addition of 40 µM HgCl2, a
still higher concentration of HgCl2, swelling was virtually
the same in the control and heat shock groups (Fig. 1C,
trace a and b, respectively). Therefore, the
threshold to induce onset of the unregulated MPT by HgCl2
was increased from 20 to 40 µM by heat shock treatment.
We also examined the effect of heat shock on MPT induction by the
amphipathic peptide mastoparan. As shown in Fig.
2A, in control mitochondria, a
low dose of mastoparan (1 µM) plus 50 µM
CaCl2 induced mitochondrial swelling that reached
half-maximum after about 15 min (trace a). This regulated
MPT induction was completely blocked by heat shock pretreatment
(trace b). When mitochondria were incubated with 3 µM mastoparan in the absence of CaCl2, rapid
swelling occurred in mitochondria from control rats (Fig.
2B, trace a) but not in mitochondria from heat
shock-treated rats (trace b). By contrast, when mitochondria
were exposed to 4 µM mastoparan, a still higher
concentration of mastoparan, the onset and progression of the MPT were
virtually same in the control and heat shock-treated groups (Fig.
2C, trace a and b, respectively), indicating that the threshold of unregulated MPT induction by mastoparan was increased by heat shock pretreatment.

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Fig. 2.
Heat shock treatment delays onset of the
regulated MPT and increases the threshold of unregulated MPT induced by
mastoparan. Mitochondria (0.5 mg of protein/ml) were added to
incubation buffer, and the onset of the MPT was monitored by
absorbance, as described under "Experimental Procedures." After a
2-3-min preincubation, 1 µM mastoparan plus 50 µM CaCl2 (A), 3 µM
mastoparan (B), 4 µM mastoparan
(C), and 200 µM CaCl2
(D) were added. Traces a and b are
control mitochondria and heat shock-treated mitochondria, respectively.
The data shown are representative of four independent
experiments.
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Mitochondria were also treated with high (200 µM)
CaCl2 alone. In mitochondria from control rats, 200 µM CaCl2 produced rapid large amplitude
swelling, which reached half-maximum within 3 min (Fig.
2D, trace a). Heat shock treatment delayed MPT
induction by high CaCl2, and half-maximum swelling after
200 µM CaCl2 required more than 20 min in
mitochondria isolated from heat shock-treated rats (Fig.
2D, trace b).
Mitochondrial Respiration, Membrane Potential, and Ca2+
Uptake after Heat Shock Treatment--
To determine whether delay of
onset of the MPT induced by heat shock was related to changes of
mitochondrial respiration, we measured mitochondrial state 3 and state
4 respiration and the respiratory control ratio using succinate plus
rotenone as substrate in control, sham control, and heat shock
mitochondria. As shown in Fig.
3A, state 3 and state 4 respiratory rates and respiratory control ratios were not significantly
different between groups. These findings indicated that the protective
effect of heat shock on MPT induction was not mediated by changes of
mitochondrial respiration.

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Fig. 3.
Heat shock treatment has no effect on
mitochondrial respiration, membrane potential, and Ca2+
uptake. A, oxygen consumption was assessed using a
Clark-type oxygen electrode in a reaction medium containing 1 mg
of protein/ml mitochondria, 150 mM sucrose, 5 mM MgCl2, 5 mM succinate, 1 µM rotenone, and 10 mM NaPi
buffer, pH 7.4. State 3 respiration rate was measured after the
addition of 200 µM ADP. B and C,
mitochondria (0.5 mg of protein/ml) were incubated in buffer containing
1 µM CsA plus 1 µM TMRM and 1 µM Fluo-5N. Red TMRM fluorescence (B) and
green Fluo-5N fluorescence (C) were measured in a
fluorescence plate reader, as described under "Experimental
Procedures." After 2-3 min of preincubation, 200 µM
CaCl2 was added to monitor Ca2+ uptake. After
complete uptake of Ca2+, 1 µM carbonyl
cyanide p-chlorophenylhydrazone (CCCP) was added
to depolarize mitochondria. Traces a (solid
lines) and b (dotted lines) are control
mitochondria and heat shock-treated mitochondria, respectively. The
data shown are representative of three independent experiments.
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To ascertain whether changes of mitochondrial membrane potential and
Ca2+ uptake were related to resistance to the MPT after
heat shock, we measured 
m and Ca2+ uptake
using the fluorescence dyes, TMRM and Fluo-5N, respectively. Mitochondria were preincubated with 1 µM TMRM, 1 µM Fluo-5N, and 1 µM CsA. CsA was added to
prevent MPT onset and consequent Ca2+ release. Under these
conditions, 
m-dependent TMRM accumulation
into mitochondria causes self-quenching of TMRM fluorescence. As shown
in Fig. 3B, during the 2-3-min preincubation, TMRM
fluorescence in both control (trace a) and heat shock
(trace b) groups was relatively low. After the addition of
200 µM CaCl2, mitochondria transiently
depolarized as Ca2+ uptake occurred, as shown by an
increase of TMRM fluorescence. Fluo-5N fluorescence increased
immediately after adding CaCl2 and then progressively
declined as uptake of Ca2+ into mitochondria occurred. Once
all the Ca2+ had accumulated, 
m recovered as
shown by the return of TMRM fluorescence to previous levels. Subsequent
addition of 1 µM CCCP to uncouple mitochondria caused
rapid mitochondrial depolarization and release of accumulated
Ca2+, as indicated by a rapid increase of TMRM and Fluo-5N
fluorescence. As shown by the representative experiments in Fig. 3,
B and C, control and heat shock mitochondria were
not different with respect to changes of 
m and rates of
Ca2+ uptake. Taken together, the results of Fig. 3 indicate
that the inhibition of MPT induction after heat shock was not due to
alterations of mitochondrial energetic status or Ca2+ accumulation.
Heat Shock Treatment Decreases ROS Formation but Has No Effect on
Glutathione--
ROS are important inducers of MPT (28, 29), and HSP
expression decreases cellular ROS formation after cytokine stimulation (7, 8). Because mitochondria are a major source of ROS production, we
measured ROS formation in isolated mitochondria loaded with 10 µM H2DCFDA. Compared with control
mitochondria, heat shock treatment deceased DCF formation rate by 27%
measured by the rate of formation of green fluorescent DCF (Fig.
4A). In control mitochondria, 1 µM CsA suppressed DCF formation by 28% to levels
nearly identical to heat shock mitochondria (Fig. 4A). By
contrast, CsA had virtually no effect on ROS formation by heat shock
mitochondria (Fig. 4A).

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Fig. 4.
Heat shock treatment decreases mitochondria
formation of reactive oxygen species but does not affect glutathione
levels. A, mitochondria (0.5 mg of protein/ml) were
incubated in a buffer containing 10 µM
H2DCFDA. Green fluorescence was monitored with a
fluorescence plate reader. After 20-30 min of incubation, the rate of
fluorescence increase became linear, and this rate was used to
represent the rate of ROS formation. Data shown are means ± S.E.
from three independent experiments (*, p < 0.01 by
Student's t test, compared with heat shock ( ) and CsA
( ) mitochondria). B, glutathione was determined in
mitochondria (5 mg of protein) and cytosolic fractions (100 µl) using
a BIOXYTECH GSH-420 kit. Data shown are means ± S.E. from four to
five independent experiments.
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Glutathione levels also modulate MPT induction, and heat shock proteins
may protect against oxidative stress by increasing the intracellular
glutathione levels (7). Accordingly, we measured mitochondrial and
cytosolic glutathione with and without heat shock. In control rats,
glutathione levels were 2.6 and 20.7 nmol/mg protein, respectively, in
mitochondria and the 100,000 × g cytosolic supernatant. However, heat shock treatment did not alter either mitochondrial or cytosolic glutathione (Fig. 4B).
Heat Shock Treatment Increases the Expression of Hsp25 but Not
Other Heat Shock Proteins in Mitochondria--
To assess the
expression of heat shock proteins, we evaluated a panel of proteins by
Western blot analysis in mitochondrial fractions from control, sham
control, and heat shock groups. As shown in Fig.
5, Hsp25 was minimal in control and sham
control mitochondria but increased substantially after heat shock. The expression of Hsp25 was also increased in cytosol (data not shown). In
contrast, the levels of the constitutively expressed mitochondrial heat
shock proteins Hsp60, Hsp70, Hsp75, and Hsp10 were not affected after
heat shock. We also assessed protein levels of putative MPT pore
proteins, CypD and VDAC, before and after heat shock. As shown in Fig.
5, CypD and VDAC expression was not altered after heat shock. To
confirm that the observed increase of Hsp25 in mitochondria was not due
to cytosolic contamination, samples were subjected to Western blot
using Hsp72 antibody. As shown in Fig. 5, Hsp72 expression was not
changed in mitochondria after heat shock but was increased in the
cytosol, indicating that the observed increase of Hsp25 expression in
mitochondria was not due to cytosolic contamination.

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Fig. 5.
Heat shock treatment increases Hsp25
expression but not Hsp60, Hsp70, Hsp10, Hsp75, CypD, or VDAC in
mitochondria. Proteins (10 µg) of liver mitochondria and cytosol
from control, sham control, and heat shock treatment groups were
resolved by 8 (Hsp75, Hsp72, Hsp60, and Hsp70), 12 (Hsp25 and VDAC), or
15% (Hsp10 and CypD) SDS-PAGE. All blots were mitochondrial proteins,
except the last blot, which were cytosolic proteins as indicated.
Western blotting was carried as described under "Experimental
Procedures." Data shown are representative of three independent
experiments.
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DISCUSSION |
Numerous studies have shown that heat shock treatment prevents
cell death in various cell types after different stimuli (1-12). The
specific mechanisms underlying heat shock protection remain unclear
(13-16, 30). Because onset of the MPT plays an important role in
regulating both necrotic and apoptotic cell death (for review see Ref.
17), this study evaluated the effect of heat shock treatment on the
MPT. We show that heat shock treatment inhibits onset of the regulated
MPT induced by low doses of HgCl2 (Fig. 1A),
mastoparan (Fig. 2A), and CaCl2 (Fig.
2D). As a result, half-maximal mitochondrial swelling
occurred more than 4 times later in the heat shock group than in the
control and sham control groups. Heat shock also increased the
threshold of the unregulated MPT induced by HgCl2 from 20 to 40 µM (Fig. 1, B and C) and by mastoparan from 3 to 4 µM (Fig. 2, B and
C). Thus, heat shock pretreatment both delays onset of the
regulated MPT and increases the threshold of the unregulated MPT. These
findings suggest that the protection by heat shock against cellular
injury may be mediated, at least in part, by preventing MPT induction.
The precise mechanisms of heat shock-induced resistance against MPT
induction remain unclear. The MPT pore is a
voltage-dependent channel (31, 32). Mitochondrial
respiration creates and maintains the mitochondrial membrane potential.
High mitochondrial membrane potential favors a closed conductance
state, whereas low membrane potential promotes pore opening.
Accordingly, we determined whether mitochondrial state 3 and state 4 respiratory rates and membrane potential were affected by heat shock
treatment. Our results showed no difference of respiratory rates,
respiratory control ratio, and membrane potential between control and
heat shock mitochondria (Fig. 3), indicating that the protection
against the MPT by heat shock was not through an alteration of
mitochondrial bioenergetic status. Ca2+ uptake into
mitochondria is also critical for induction of the regulated MPT (33),
but mitochondrial Ca2+ uptake was also unaffected by heat
shock treatment (Fig. 3C). These results are consistent with
previous studies (4, 15) showing that heat shock treatment itself does
not change mitochondrial respiration or membrane potential.
In previous studies, heat shock protection against apoptotic stimuli
was associated with decreased ROS production and increased intracellular glutathione (7, 34). By using H2DCFDA as a probe of ROS formation, we found that ROS production by mitochondria was decreased by 27% in heat shock mitochondria (Fig. 4A),
although both mitochondrial and cytosolic levels of glutathione were
unaffected (Fig. 4B). The mechanism by which heat shock
treatment causes this modest decrease of ROS production is unclear. One
possibility is that heat shock treatment blocks transient MPT pore
openings (35, 36) or sustained pore openings in a small subpopulation of mitochondria. Transient pore openings or the occurrence of the MPT
in a small mitochondrial subpopulation may contribute to ROS formation
in control mitochondria. This hypothesis is consistent with the finding
that incubation of control mitochondria with 1 µM CsA
decreased ROS formation to the levels observed in heat shock
mitochondria (Fig. 4A). Inhibition of the MPT may also be the basis by which heat shock prevents
H2O2-induced decreases in mitochondrial state 3 respiration and membrane potential reported by others (4).
By using Western blot analysis, we found that among the mitochondrial
HSPs and putative MPT complex proteins evaluated, only Hsp25 increased
after heat shock treatment. Other mitochondrial HSPs (Hsp10, Hsp60,
Hsp70, and Hsp75) and the putative MPT complex proteins CypD and VDAC
were not affected (Fig. 5). Hsp25 belongs to a family of small heat
shock proteins, including Hsp27 (human homologue of Hsp25) and
B-crystalline (37). Hsp25 was reported previously (38) to be in the
cytosol and nucleus. Recently, Hsp25 was identified in the mitochondria
of mouse PC12 cells (39) and heat-shocked Jurkat T-lymphocytes (40). In
our experiments, Hsp25 was barely detectable in liver mitochondria from
control rats but increased greatly in both mitochondrial (Fig. 5) and cytosolic fractions (data not shown) after heat shock.
Hsp25 may regulate MPT pore opening in two ways. First, Hsp25
expression may suppress ROS production and prevent adverse consequences such as mitochondrial depolarization, cytochrome c release,
and apoptosis. Overexpression of human Hsp27 and mouse Hsp25 protects mouse L929 fibroblasts against oxidative stress induced by
H2O2 and increases the activity of
glucose-6-phosphate dehydrogenase, a regulator of antioxidant pathways
(34). Inhibition of Hsp27 expression with hsp27 antisense
also potentiates mitochondrial depolarization and cytochrome
c release after apoptotic stimuli in Jurkat cells (40). In
mice lacking heat shock transcription factor 1, cardiac Hsp25
expression is decreased; superoxide production is increased;
mitochondrial proteins including adenine nucleotide translocator 1 are
more oxidized, and isolated mitochondria are more sensitive to MPT
induction by CaCl2 (41). In our experiments, however, ROS
formation declined only 27% after heat shock, which was
disproportionate with the suppression of MPT induction. In addition,
this decrease of ROS formation appeared to be the consequence of MPT
suppression rather than its cause, because CsA treatment of control
mitochondria decreased ROS production to that observed in heat shock
mitochondria (Fig. 4A).
As a second mechanism, Hsp25 may exert direct chaperone-related
regulation of pore conductance, as proposed by a recent model of pore
formation and gating (20). In this model, stress induces misfolding of
mitochondrial membrane proteins, which expose their hydrophilic
residues to the bilayer phase and then cluster to form aqueous pores
that are permeable to ions and molecules up to a molecular mass of
about 1,500 Da. Chaperone proteins and CypD bind to the pore complex to
shut off conductance. However, increased Ca2+ causes CypD
to perturb the MPT pore complex to an open conductance state, an
effect that is antagonized by CsA. As the number of misfolded protein
clusters exceeds the number of chaperones available to block MPT pore
conductance, unregulated pores accumulate. This model explains
observations that low levels of various MPT inducers cause
Ca2+-dependent and CsA-sensitive
permeabilization of the inner membrane, whereas high levels cause
Ca2+-independent and CsA-insensitive onset of the MPT
(20-24). The association of increased Hsp25 expression with inhibition
of the regulated MPT and an increased threshold for the unregulated MPT implicates Hsp25 as a candidate chaperone involved in MPT regulation as
predicted by the model. The new model is also consistent with the
recent finding that overexpression of mitochondrially targeted CypD
desensitizes cells against apoptotic stimuli (42).
Our findings in the present study that heat shock treatment inhibits
onset of the regulated MPT and increases the threshold for onset of the
unregulated MPT (Figs. 1 and 2) can be explained by the model of
chaperone regulation of the MPT pore. First, increased concentration of
heat-inducible chaperones (e.g. Hsp25) might be expected to
retard onset of the MPT by a mass action effect, because the
physiological role of the chaperones is to prevent mitochondrial
permeabilization and uncoupling after formation of clusters of
misfolded mitochondrial membrane proteins. Second, increased
availability of heat-induced chaperones would be expected to block a
greater number of the misfolded protein clusters, which explains the
increased threshold for unregulated MPT induction by both
HgCl2 and mastoparan. In this respect we found that the concentration of inductor needed for onset of the unregulated MPT
increased from 20 µM HgCl2 and 3 µM mastoparan for control mitochondria to 40 µM HgCl2 and 4 µM mastoparan in
heat shock mitochondria (Figs. 1C and 2C).
The increased threshold of unregulated MPT induction is thus explained
by the proposed model.
In summary, this study shows for the first time that heat shock
pretreatment delays onset of the regulated MPT and increases the
threshold of unregulated MPT induction. Heat shock treatment does not
alter mitochondrial respiration, membrane potential, Ca2+
uptake, or mitochondrial and cytosolic glutathione levels but does
cause a modest decrease of ROS production. Of the several HSPs
examined, only Hsp25 expression increased after heat shock. Thus, the
effect of heat shock on MPT inhibition may be partially through the
up-regulation of mitochondrial Hsp25 expression. The effects of heat
shock treatment on MPT induction may underlie the mechanism of heat
shock protection against various different cell injuries.