Regulation of the mitochondrial permeability transition by
matrix Ca2+ and voltage during anoxia/reoxygenation
Paavo
Korge,
Henry M.
Honda, and
James N.
Weiss
Cardiovascular Research Laboratory, Departments of Medicine
(Cardiology) and Physiology, University of California at Los Angeles
School of Medicine, Los Angeles, California 90095
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ABSTRACT |
We
studied the interplay between matrix Ca2+ concentration
([Ca2+]) and mitochondrial membrane potential
(
) in regulation of the mitochondrial permeability transition
(MPT) during anoxia and reoxygenation. Without Ca2+
loading, anoxia caused near-synchronous 
dissipation,
mitochondrial Ca2+ efflux, and matrix volume shrinkage when
a critically low PO2 was reached, which was
rapidly reversible upon reoxygenation. These changes were related to
electron transport inhibition, not MPT. Cyclosporin A-sensitive MPT did
occur when extramitochondrial [Ca2+] was increased to
promote significant Ca2+ uptake during anoxia, depending on
the Ca2+ load size and ability to maintain 
. However,
when [Ca2+] was increased after complete 
dissipation, MPT did not occur until reoxygenation, at which time
reactivation of electron transport led to partial 
regeneration.
In the setting of elevated extramitochondrial Ca2+, this
enhanced matrix Ca2+ uptake while promoting MPT because of
less than full recovery of 
. The interplay between 
and
matrix [Ca2+] in accelerating or inhibiting MPT during
anoxia/reoxygenation has implications for preventing reoxygenation
injury associated with MPT.
cardiomyocytes; mitochondrial Ca2+ uptake; Ca2+ efflux; permeability transition pore
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INTRODUCTION |
MYOCARDIAL VIABILITY
AFTER reperfusion/reoxygenation is critically dependent on the
recovery of mitochondrial membrane potential (
) to provide the
driving force for oxidative phoshorylation. A major factor that could
limit recovery of 
on reperfusion/reoxygenation is
Ca2+-dependent opening of large nonselective permeability
transition pores (PTP) in the inner mitochondrial membrane, an event
called mitochondrial permeability transition (MPT) (6,
19). In addition to triggering apoptotic signaling, MPT
occurring near synchronously in the majority of mitochondria in a cell
leaves ATP production capability inadequate and facilitates necrotic
cell death. Preventing MPT during reperfusion/reoxygenation has obvious
therapeutic promise, but developing effective strategies will require
better understanding of its regulation under these conditions. For
example, cyclosporin A (CSA) is the most potent inhibitor of MPT known,
and concentrations <400 nM partially prevented
reperfusion/reoxygenation injury. Higher concentrations, however,
exacerbated damage (15, 30). As emphasized by Bernardi
(2), CSA's ability to block PTP opening is variable
because it is not a completely specific inhibitor and its effect can be
antagonized by increased matrix Ca2+ concentration
([Ca2+]), oxidative stress, and other factors known to
occur during reperfusion/reoxygenation. Therefore, additional
strategies to enhance the efficacy of direct PTP blockers would be
highly desirable.
PTP open-closed transitions underlying MPT are highly regulated
(2). Matrix Ca2+ is the most important
regulatory factor, but there are multiple modulators, including 
(2). Bernardi et al. (5) have shown that
following accumulation of a small Ca2+ load by respiring
mitochondria, which by itself was insufficient to induce MPT, 
dissipation by the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) rapidly promoted
PTP opening. PTP thus behaved as a voltage-dependent channel with higher open probability at depolarized membrane potentials. The idea of
a voltage sensor regulating PTP opening is attractive because it could
provide a common mechanism to account for the actions of a variety of
PTP modulators: activators may shift the threshold voltage to more
negative 
and cause PTP opening at values close to physiological

, with inhibitors doing the converse (3).
However, mitochondrial Ca2+ uptake is also regulated by

, which provides the driving force for Ca2+ uptake
into the matrix through the Ca2+ uniporter. Well-maintained

promotes Ca2+ uptake when cytoplasmic free
[Ca2+] increases, as occurs during ischemia and
hypoxia (6). Therefore, during ischemia/hypoxia,
maintenance of 
is predicted to have dual effects: it should
promote PTP opening by increasing mitochondrial Ca2+
uptake into the matrix, but inhibit PTP opening by the
voltage-dependent mechanism. Conversely, although 
dissipation
during ischemia/hypoxia protects the mitochondrial matrix from
further Ca2+ accumulation, its sudden dissipation during
hypoxia/ischemia may promote MPT if mitochondria are already
Ca2+ loaded. In this study, we investigated the interplay
between these opposing effects of 
by examining the effects of
anoxia/reoxygenation on PTP opening in suspensions of isolated cardiac
mitochondria subject to varying Ca2+ loads as well as in
permeabilized and intact myoctyes.
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MATERIALS AND METHODS |
Isolation of mitochondria.
Mitochondria were isolated from adult rabbit hearts by enzymatic
digestion of finely minced tissue with the bacterial protease nagarase
(0.5 mg/ml) for 10 min on ice in homogenization buffer (250 mM sucrose,
1 mM EGTA, and 10 mM MOPS, pH 7.4 with Tris) followed by differential
centrifugation, as described previously (25). Mitochondria
were resuspended in the EGTA-free homogenization buffer to give about
40 mg mitochondrial protein per milliliter, kept on ice, and used
within 6 h after isolation. Respiratory control ratio was
regularly determined in mitochondrial incubation buffer [120 mM KCl,
10 mM HEPES (pH 7.4), and 2 mM potassium phosphate] after consecutive
addition of 2.5 mM pyruvate, malate, and glutamate and 0.5 mM ADP. Only
mitochondrial preparations with ratio >5 were used in these experiments.
Isolation and permeabilization of myocytes.
Ventricular myocytes were isolated from adult rabbit hearts by
conventional enzymatic methods described previously (14). Myocytes were stored in normal Tyrode solution and used within 5-6
h. In some experiments, the sarcolemma was permeabilized by treating
cells with digitonin (20 µM) in a buffer that contained (in mM) 135 KCl, 1 MgCl2, 3 ATP, 0.5 EGTA, and 10 HEPES (pH 7.2) for 10 min. Myocytes were pelleted at 50 g, washed, and resuspended in the same buffer as described (1). After this treatment, myocytes retained a rod-shaped morphology, with intact and functional sarcoplasmic reticulum, contractile elements, and mitochondria (1).
Experimental conditions for anoxia/reoxygenation.
All experiments were carried out with a spectrofluorometer (Ocean
Optics) in a closed cuvette at room temperature (22-24°C). Mitochondria or isolated cells were made anoxic by injecting a stream
of nitrogen through the hole in the cuvette cover against the surface
of the buffer (2 ml) so that the stirred buffer had no contact with the
air. Reoxygenation was accomplished by substituting nitrogen with
oxygen (95% O2-5% CO2) in the stream. A
fiber-optic oxygen sensor was inserted through the same hole, and
partial pressure of oxygen (PO2) in the buffer
was continuously recorded. The rate of
PO2 decrease (the slope of O2 trace
in Fig. 1) was dependent on the flow rate
of the nitrogen stream and was set to achieve critically low
PO2 in ~7-8 min, unless indicated
otherwise.

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Fig. 1.
Effects of anoxia on mitochondrial membrane potential
( ) in a non-Ca2+-loaded mitochondrial suspension.
A: simultaneous recording of buffer
PO2 and mitochondrial  . Between the
arrows, the surface of stirred buffer was equilibrated with
N2 in place of O2 as indicated. At a critically
low PO2, near-synchronous  dissipation
occurred, which reversed promptly upon reoxygenation. B:
same as A, except that after hypoxia, the N2
stream was shut off, allowing the buffer to reequilibrate slowly with
air. Full  recovery occurs at lower PO2
than the O2 electrode can detect. Mitochondria (1.0 mg)
were suspended in incubation media (2 ml) that contained (in mM) 120 KCl, 1 Pi, 10 HEPES, and 0.0004 tetramethylrhodamine methyl
ester (TMRM), pH 7.4. Mitochondria were energized with 5 mM pyruvate,
malate, and glutamate (A), representative tracing from 6 different preparations; mitochondria were energized with 5 mM succinate
(B), representative tracing from 6 different preparations.
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Determination of mitochondrial matrix volume and 
.
Mitochondria (0.5-1.0 mg) were added to 2 ml of mitochondrial
incubation buffer that contained 400 nM tetramethylrhodamine methyl
ester (TMRM) and continuously stirred. Changes in mitochondrial matrix
volume were estimated by measuring 90° light scattering using the
Ocean Optics spectrofluorometer with excitation and emission
wavelengths set at 520 nm, similar to the method described by Haworth
and Hunter (20). Changes in matrix volume are reported as
a percentage of maximum (100%) swelling induced by adding 2.5 µg of
alamethicin at the end of the experiment. 
was estimated by transmembrane distribution of TMRM. Changes in 
are expressed as percentage of the TMRM fluorescence level at 580 nm in the presence
of coupled mitochondria and substrates (0%), relative to the
fluorescence after addition of 0.5 µM FCCP to fully depolarize mitochondria (100%). Similar extent of 
dissipation was observed after treatment of energized mitochondria with 1 mM cyanide or 10 µM
antimyosin A. Both light scattering and TMRM fluorescence emission were
recorded simultaneously with PO2.
Determination of mitochondrial 
in isolated intact and
permeabilized cells.
Isolated intact or permeabilized cells were suspended in a buffer that
contained (in mM) 135 KCl, 1 MgCl2, 0.5 EGTA, 10 HEPES (pH
7.2), 5 malate-Tris, 5 glutamate-Tris, 5 succinate-Tris, and 0.0008 TMRM. In experiments with permeabilized cells, the buffer also
contained 3.5 mM ATP. Mitochondrial membrane potential-dependent uptake
of TMRM was established by fluorescence change resulting from
dissipation of 
with 0.5 µM FCCP. In intact cells, 
was also rapidly and completely dissipated with other mitochondrial inhibitors (NaCN and antimyosin A). In experiments with anoxia, buffer
PO2 was measured simultaneously with TMRM
fluorescence emission.
Mitochondrial Ca2+ uptake and
efflux determination.
Mitochondria (0.5-1 mg/ml) were incubated in the buffer described
above that contained 1 µM Calcium Green-5N (salt form). The
suspension was continuously stirred in the fluorometer cuvette, and
changes in extramitochondrial [Ca2+] were followed by
recording of Calcium Green fluorescence (excitation/emission, 475/515
nm). Calibration was achieved by adding known amounts of
Ca2+ to the buffer as described previously
(23), but in the presence of mitochondria and 5 µM
ruthenium red to block Ca2+ uptake.
Other assays and chemicals.
Mitochondrial protein was determined by the Lowry method. CSA was a
generous gift of Ciba-Geigy. Percoll was purchased from Pharmacia and
fluorescent dyes from Molecular Probes. All other chemicals were
purchased from Sigma. Mitochondrial substrates were free acids adjusted
to buffer pH with Tris.
All results are presented as original tracings with indication of the
number of different preparations used to reproduce findings. 
recovery on reoxygenation for different groups is presented as
means ± SD.
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RESULTS |
Effects of anoxia/reoxygenation in mitochondria without
Ca2+ loading.
Figure 1A illustrates oxygen content and mitochondrial

in a mitochondrial suspension as a nitrogen (N2)
stream was directed against buffer surface in a closed cuvette. 
remained constant until PO2 reached the
threshold of electrode sensitivity, at which point rapid and complete
depolarization occurred (n = 6 preparations). On
replacing N2 with O2, electron transport
resumed and restored 
rapidly as PO2
increased (n = 6 preparations). If, instead, the
N2 stream was discontinued to let room air reoxygenate the suspension, electron transport resumed and restored 
well before the threshold of the oxygen electrode was reached, consistent with the
high-affinity binding of oxygen to cytochrome oxidase (Fig.
1B). Under these conditions, the ability of mitochondria to
restore 
during reoxygenation depended on the presence of substrates. In Fig. 1A, mitochondria were energized with
pyruvate, malate, and glutamate, but each of these substrates
individually, as well as succinate, were also effective (e.g., Fig.
1B with succinate alone). Importantly, when no
extramitochondrial Ca2+ was added, 
dissipation
during anoxia was always rapid, occurring synchronously in the whole
population of mitochondria (n = 6 preparations). Furthermore, reoxygenation always resulted in almost full recovery of

, irrespective of the substrates used or whether a critical level
of PO2 was achieved rapidly or slowly. Figure
2 summarizes 
recovery upon
reoxygenation in the presence of different substrates; recovery with
succinate or pyruvate, malate, and glutamate was close to 100%, but
slightly lower in the presence of pyruvate alone.

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Fig. 2.
Recovery of  upon reoxygenation, with different
substrates, extramitochondrial Ca2+ concentration
([Ca2+]) loads, and  depolarization (DP). 
value was measured 2-3 min after reoxygenation and expressed as a
percentage, with 100% corresponding to energized mitochondria and 0%
to carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone
(FCCP)-treated mitochondria. Mitochondria were incubated before
reoxygenation with 5 mM succinate (suc), 5 mM pyruvate (pyr), 5 mM each
of pyruvate, malate (mal), and glutamate (glut), succinate + low
(35 ± 9 µM) Ca2+, pyruvate + low (10 ± 4 µM) Ca2+, succinate + high (60 ± 16 µM)
Ca2+, pyruvate + 350 nM cyclosporin A (CSA) + low
(15 ± 5 µM) Ca2+, succinate + high (50 µM)
Ca2+ added after anoxia-induced depolarization, and
succinate + 350 nM CSA + high (50 µM) Ca2+
added after anoxia-induced depolarization. Numbers above each bar
represent the number of separate determinations, with the number of
different mitochondrial preparations indicated in parentheses.
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dissipation during anoxia was accompanied by slight matrix
shrinkage, which reversed with 
recovery upon reoxygenation (n = 3 preparations, Fig.
3A, bottom trace).

dissipation during anoxia also resulted in Ca2+
efflux (n = 5 preparations, Fig. 3B). From
the initial part of the tracing, it is evident that freshly isolated
energized mitochondria accumulated contaminant Ca2+ from
the buffer. During anoxia, mitochondria released this Ca2+
(~1 µM per milligram of protein) when 
dissipation occurred. Ca2+ efflux was attributed to the Ca2+
uniporter, since upon reoxygenation, mitochondria rapidly reaccumulated this Ca2+ (Fig. 3B), which would not have been
possible had PTP opening occurred. Also, blocking Ca2+
transport by the Ca2+ uniporter with 5 µM ruthenium red
completely prevented Ca2+ efflux when 
dissipation
occurred during anoxia (data not shown).

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Fig. 3.
Effects of anoxia/reoxygenation on  , matrix volume
(A), and extramitochondrial [Ca2+]
(B) in non-Ca2+-loaded mitochondria.
A: simultaneous recordings of mitochondrial  and
matrix volume during anoxia (N2 arrow) and reoxygenation
(O2 arrow). Matrix volume shrank with  dissipation
and recovered with reoxygenation. After reoxygenation, addition of 0.5 mM EGTA had little effect on  or matrix volume. At the end of the
trace, FCCP (0.5 µM) and alamethicin (Ala; 2.5 µg) were added to
calibrate changes in  and matrix volume. Mitochondria were
suspended at 0.5 mg/ml in incubation buffer that contained 5 mM
pyruvate. Traces are representative of 6 different preparations.
B: mitochondrial matrix [Ca2+] during
anoxia/reoxygenation. Hypoxia induced rapid Ca2+ efflux
from mitochondria (1 mg/ml) that coincided temporally with 
dissipation. Upon reoxygenation, mitochondria reaccumulated
Ca2+ coincident with  recovery. At the end of the
trace, Ca2+ pulses (1 µM each) were added after
mitochondrial uptake was blocked by 5 µM ruthenium red (RR),
indicating that release/uptake by mitochondria was ~2 µM.
Incubation buffer as in Fig. 1B, except 1 µM Calcium
Green-5N replaced TMRM (5 preparations).
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Effects of Ca2+-loading mitochondria
before 
dissipation during anoxia.
When extramitochondrial Ca2+ was increased by adding small
Ca2+ pulses before 
dissipation during anoxia, a
different response was observed. With pyruvate as the substrate, only a
few 5-µM Ca2+ pulses were required to accelerate 
dissipation in a subpopulation of mitochondria before
PO2 reached the critical level inhibiting electron transport, whereupon 
rapidly dissipated throughout the
full population (n = 6 preparations, Figs. 2 and
4A). In contrast to the matrix
shrinkage observed in the absence of added Ca2+ (Fig.
3A), matrix swelling consistent with MPT now occurred in parallel with 
dissipation (n = 6 preparations,
Fig. 4A). Upon reoxygenation, there was partial, transient
recovery of 
, followed by 
dissipation and further matrix
swelling. These changes were due to Ca2+-induced MPT, since
they were prevented by 350 nM CSA (n = 3 preparations, Figs. 2 and 4B). With CSA present, 
dissipation and
matrix volume changes were similar to those in the absence of added
extramitochondrial Ca2+ (Fig. 3A), despite a
greater number of Ca2+ pulses.

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Fig. 4.
Ca2+-loading mitochondria before 
dissipation during anoxia: effects on  and matrix volume.
A: in 5 mM pyruvate-energized mitochondria (0.3 mg/ml), 2 Ca2+ pulses (5 µM, arrows) added during anoxia
(N2 arrow) before  dissipation caused initially slow
 depolarization and matrix swelling consistent with mitochondrial
permeability transition (MPT) in a subpopulation of the mitochondria,
followed by near-synchronous rapid  dissipation and further
matrix swelling in the remainder. Reoxygenation (O2 arrow)
led to transient partial  recovery, followed by 
dissipation and further matrix swelling. Addition of both EGTA and
succinate were required for recovery of  and matrix volume (3 preparations). B: same as A, except in the
presence of 350 nM CSA. Addition of 5-µM Ca2+ pulses now
failed to induce slow  depolarization or matrix swelling,
although near-synchronous  dissipation still occurred at
critically low PO2. Changes reversed rapidly
with reoxygenation alone, indicating that MPT had been prevented
(confirmed in 3 preparations).
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However, other substrates were more protective against
Ca2+-induced MPT. When pyruvate was added together
with malate and glutamate (n = 2), or when succinate
was used instead of pyruvate (n = 4), a larger number
of Ca2+ pulses failed to initiate MPT during anoxia (Fig.
5A). With larger Ca2+ loads (n = 4 preparations), however,
matrix swelling that indicated MPT (Fig. 5B) occurred when

dissipated abruptly during anoxia, favoring MPT by way of inner
membrane depolarization. The increase in extramitochondrial
[Ca2+] during anoxia decreased 
recovery on
reoxygenation in succinate-energized mitochondria (n = 4 preparations, Fig. 2). Finally, with more gradual onset of 
dissipation during partial anoxia, a similar pattern of slow partial

depolarization associated with matrix swelling, as in
pyruvate-energized mitochondria, was observed (n = 3 preparations, Fig. 5C, compared with Fig. 4A).
The superiority of succinate at delaying MPT during hypoxia in the
face of increasing extramitochondrial Ca2+ is
potentially explained by the proposed role of complex I electron flow
in the mechanism of PTP opening (10). The mechanism by which the addition of malate and glutamate to pyruvate protected against Ca2+-induced MPT during anoxia is less clear, but
could be related to prevention of complex I dysfunction, thought to be
responsible for diminished 
in kidney cells during
hypoxia/reoxygenation (32).

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Fig. 5.
Ca2+-loading mitochondria before 
dissipation during anoxia: substrate dependence and reversal of MPT by
Ca2+ removal + CSA. A: with succinate in
place of pyruvate, five 5-µM Ca2+ pulses were still
insufficient to trigger slow  depolarization and matrix swelling
indicative of MPT (4 preparations). B: six 10 µM
Ca2+ pulses added before and during anoxia did not induce a
slow phase of  dissipation or matrix swelling indicating MPT.
When critically low PO2 caused rapid 
dissipation, however, matrix swelling indicating MPT now occurred,
which was exacerbated by reoxygenation (O2). After
reoxygenation, addition of EGTA and 350 nM CSA led to recovery of
 and reversal of matrix swelling (5 preparations). C:
similar experiment in which anoxia was created more slowly by
decreasing the flow of the N2 stream. Five 15 µM
Ca2+ pulses induced slow  dissipation and matrix
swelling during anoxia indicating MPT, as in pyruvate-energized
mitochondria (Fig. 3A). After reoxygenation, addition of 0.5 mM EGTA alone did not reverse MPT, whereas addition of 350 nM CSA after
EGTA led to recovery of  and reversal of matrix swelling (3 preparations). A-C: mitochondria (0.3 mg/ml) were
energized with 5 mM succinate, and  was recorded with TMRM.
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Figure 6 shows that compared with their
preanoxic state, mitochondria became sensitized to
Ca2+-induced MPT during reoxygenation. In Fig.
6A, two Ca2+ pulses administered during anoxia
led to Ca2+ efflux when 
dissipated, but
Ca2+ reuptake occurred upon reoxygenation. This net
Ca2+ accumulation was maintained at least for >5 min
without any indication of Ca2+ efflux, until FCCP was added
to rapidly dissipate 
and release the accumulated
Ca2+. In Fig. 6B, four Ca2+ pulses
administered before anoxia were also readily accumulated. During
anoxia, Ca2+ efflux again occurred coincident with 
dissipation. Upon reoxygenation, however, Ca2+ reuptake was
now only transient and turned into Ca2+ efflux within a few
minutes. This was due to MPT, since addition of CSA terminated
Ca2+ efflux and allowed the mitochondria to
reaccumulate the released Ca2+. Similar findings were
obtained in two other preparations. Thus a Ca2+ load that
was well tolerated by fully polarized mitochondria before anoxia was
now capable of triggering MPT during reoxygenation. MPT in this setting
was most likely precipitated by rapid Ca2+ uptake at the
start of reoxygenation before 
had recovered fully. As shown in
Figs. 4A and 5B, 
recovery in this
situation was only partial, which lowers the threshold for MPT at a
given level of matrix Ca2+. Figure
7A shows that pretreatment
with CSA (350 nM) also prevented the majority of anoxia-induced
Ca2+ efflux from Ca2+-loaded mitochondria. The
small component of Ca2+ efflux that remained was blocked by
ruthenium red (n = 2 preparations), indicating that it
originated from reversal of the Ca2+ uniporter with 
dissipation. However, CSA was often unable to restore 
to
allow Ca2+ reuptake (Fig. 7B) and reverse matrix
swelling after anoxia-induced MPT unless the extramitochondrial
Ca2+ was removed with EGTA (Fig. 5, B and
C). Thus PTP opening during anoxia/reoxygenation could
generally be reversed by CSA and Ca2+
removal.

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Fig. 6.
Ca2+-loading mitochondria before 
dissipation: effects on mitochondrial Ca2+ fluxes.
A: a small Ca2+ load of two 15 µM
Ca2+ pulses during anoxia insufficient to induce MPT
resulted in mitochondrial Ca2+ efflux, coinciding with
 dissipation that reversed spontaneously upon reoxygenation and
was reproduced by  dissipation with FCCP (3 preparations).
B: four 10 µM Ca2+ pulses administered before
the onset of anoxia (N2) were readily accumulated by
mitochondria, which subsequently released the accumulated
Ca2+ as a result of anoxia-induced  dissipation. On
reoxygenation, mitochondria initially reaccumulated Ca2+
coincident with  recovery. However, Ca2+ uptake soon
turned into Ca2+ efflux due to MPT, reversed by subsequent
application of 350 nM CSA (2 preparations). In both A and
B, mitochondria (0.4 mg/ml) were energized with 5 mM
succinate, and changes in extramitochondrial [Ca2+]
recorded with 1 µM Calcium Green-5N.
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Fig. 7.
Ca2+-loading mitochondria before 
dissipation during anoxia: effects of CSA on mitochondrial
Ca2+ fluxes. A: in the presence of 350 µM CSA,
three 15 µM Ca2+ pulses administered during anoxia caused
modest Ca2+ efflux during anoxia, which was blocked by
ruthenium red (2 µM), indicating involvement of the Ca2+
uniporter (3 preparations). B: four 15 µM Ca2+
pulses initiated Ca2+ efflux during anoxia
(N2), which was not reversed by reoxygenation even after
350 nM CSA. FCCP (1 µM) also had no effect, indicating that
mitochondria were fully depolarized (3 preparations). In both
A and B, mitochondria (0.4 mg/ml) were energized
with 5 mM succinate, and changes in extramitochondrial
[Ca2+] recorded with 1 µM Calcium Green-5N.
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Effects of Ca2+ loading after 
dissipation during anoxia.
In the previous experiments, extramitochondrial Ca2+ was
added before 
dissipated during anoxia. During anoxia in intact
cardiac myocytes, however, Ca2+ may continue to rise after

collapses (8), generating a possible situation in
which rapid 
recovery during reoxygenation could result in
significant Ca2+ uptake and trigger MPT. The resulting

dissipation and Ca2+ release from the so-affected
mitochondria might then enhance the probability of MPT in the
remainder. This scenario is demonstrated in Fig.
8A, in which addition of
Ca2+ pulses after 
dissipation had no significant
effect on matrix volume, indicating that MPT had not occurred
(n = 5 preparations). Also consistent with the absence
of MPT, upon reoxygenation, 
began to recover. However, the
recovery was transient and followed by rapid depolarization and matrix
swelling, indicating Ca2+-dependent MPT (n = 5 preparations). Both 
and matrix volume recovered after
addition of EGTA and CSA, confirming that changes were Ca2+
dependent and CSA sensitive (n = 5 preparations). Thus

recovery on reoxygenation seemed to depend on PTP open-closed
conformation (Figs. 2, 4, 5, and 8).

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Fig. 8.
Effects of Ca2+-loading mitochondria after
 dissipation during anoxia. A: two 25 µM
Ca2+ pulses administered after  dissipation during
anoxia (N2) had no further effect on  or matrix
volume. Upon reoxygenation,  recovery was partial and transient,
followed by  dissipation and matrix swelling indicating MPT that
reversed with EGTA (0.5 mM) and CSA (350 nM; 5 preparations).
B: eight 10 µM Ca2+ pulses administered after
 dissipation during anoxia (N2) led to no
Ca2+ accumulation until reoxygenation. With reoxygenation,
transient Ca2+ accumulation was followed by
Ca2+ efflux partly reversed by CSA (350 nM; 3 preparations). Mitochondria (0.5 mg/ml) were energized with 5 mM
succinate; extramitochondrial Ca2+ and  recorded with
Calcium Green-5N and TMRM, respectively.
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Figure 8B shows that no Ca2+ uptake occurred
when 10-µM Ca2+ pulses were added after 
was
already fully dissipated during anoxia (n = 3 preparations), as expected from the lack of driving force for the
Ca2+ uniporter. Subsequent reoxygenation, however,
promoted Ca2+ uptake, which rapidly turned into net
Ca2+ efflux. CSA reversed this Ca2+ efflux into
accumulation, indicating that PTP opening was responsible (Fig.
8B).
MPT induced by rapid 
regeneration in the presence of high
extramitochondrial [Ca2+] could also be demonstrated
by dissipating 
with rotenone instead of anoxia before adding
Ca2+ pulses (n = 5 preparations, Fig.
9A). Mitochondria were then energized with succinate (a substrate that enters distally to the
complex 1 inhibition site of rotenone). Succinate regenerated 
leading to Ca2+ uptake, but this soon turned into net
Ca2+ efflux (Fig. 9A) unless CSA was present to
inhibit MPT (Fig. 9B). In the latter case, the potent
chemical MPT inducer phenylarsine oxide could still induce MPT.

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Fig. 9.
Effects of rapid  recovery on mitochondrial
Ca2+ uptake and subsequent Ca2+ efflux through
permeability transition pores. Mitochondria (1 mg) were incubated with
5 µM rotenone (Rot) that rapidly and completely dissipated 
(not shown). Ca2+ pulses (25 µM) were added as indicated,
and then mitochondria were reenergized by adding 5 mM succinate.
Transient Ca2+ uptake was followed by Ca2+
efflux (A), which was prevented by pretreatment with 350 nM
CSA (B). In the latter case, 25 µM phenylarsine oxide
(PAO) induced Ca2+ efflux despite CSA. Changes in
extramitochondrial [Ca2+] were recorded by Calcium
Green-5N (5 preparations).
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Effect of anoxia/reoxygenation on mitochondrial

in intact or permeabilized myocytes.
To determine whether the responses of in situ mitochondria to
anoxia/reoxygenation were similar to those of isolated mitochondria, we
performed analogous experiments in intact and permeabilized myocytes,
using TMRM to estimate 
(Fig.
10). Figure
10A shows that when intact myocytes were added to
low-Ca2+ KCl buffer (see MATERIALS AND METHODS)
containing mitochondrial substrates (succinate + malate + glutamate), there was a significant uptake of TMRM (Fig.
10A). After turning on the N2 stream, buffer PO2 declined progressively, and when the
threshold of O2 electrode sensitivity was reached, 
rapidly and synchronously dissipated. Turning off the N2
stream led to slow recovery of 
that dissipated rapidly when
N2 was turned back on. Subsequent reoxygenation with an
O2 stream resulted in immediate recovery of 
(Fig.
10A). Similar results were obtained in five preparations.
These responses were virtually identical to those in isolated
mitochondria in the absence of Ca2+ load (Fig. 1).

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|
Fig. 10.
The effects of anoxia/reoxygenation on in situ
mitochondria are similar to isolated mitochondria. A:
isolated myocytes were suspended in a KCl buffer (see MATERIALS
AND METHODS) that contained mitochondrial substrates (5 mM of
malate, glutamate, and succinate) and 0.8 µM TMRM. Final KCl buffer
also contained 13 mM Na+ added with a 200-µl suspension
of myocytes. With anoxia (N2),  (top
trace) dissipated rapidly and synchronously when critical
PO2 was reached (bottom trace) and
rapidly recovered upon reoxgenation (O2). Subsequent
application of FCCP shows that  dissipation during anoxia had
been complete (5 preparations). B: digitonin-permeabilized
cells incubated in KCl buffer that contained 3.5 mM ATP and
mitochondrial substrates with TMRM as above. After critical
PO2 was reached (bottom trace),
 (top trace) rapidly decreased, but incompletely.
Subsequent addition of 10 µM oligomycin (Oligo) caused additional
 dissipation that reversed with reoxygenation (see text for
explanation) (3 preparations). C: isolated mitochondria
incubated in KCl buffer with 3.5 mM ATP and mitochondrial substrates
under the same conditions as in B showed the same response.
Mitochondrial volume changes (middle trace) are also shown.
 dissipation after oligomycin was complete as demonstrated with
addition of 0.5 µM FCCP at the end of experiment (3 preparations).
N2 out refers to discontinuing the N2 stream,
O2 to starting the O2 stream.
|
|
A similar picture was observed in digitonin-permeabilized cells
(n = 4 preparations, Fig. 10B).
Permeabilized cells added to KCl buffer that contained mitochondrial
substrates and 3.5 mM MgATP accumulated TMRM. During anoxia, they
maintained 
until PO2 reached a critical
level, at which point 
dissipated rapidly and synchronously, but
to an incomplete extent. The partial 
dissipation was due to the
presence of MgATP, which allowed ATP synthase (in reverse mode) to pump
enough protons to maintain a partial proton gradient, since inhibition
of ATP synthase with oligomycin led to rapid and complete collapse of

(n = 3 preparations). Identical findings were
obtained in isolated mitochondria when the incubation media contained
substrates and MgATP during anoxia/reoxygenation (Fig. 10C,
n = 3 preparations).
 |
DISCUSSION |
MPT, due to PTP opening, is widely recognized as having an
important role in both apoptotic and necrotic cell death
(6). PTP opening is regulated by multiple factors, among
which matrix [Ca2+] and mitochondrial 
are two of
the most crucial. Elevated mitochondrial matrix [Ca2+] is
regarded as the most essential requirement, and 
dissipation significantly enhances PTP open probability by decreasing the [Ca2+] required to induce MPT. During
hypoxia/reoxygenation, both occur. Furthermore, cell recovery upon
reoxygenation has been shown to depend on recovery of mitochondrial

(7), and recovery is unlikely once critical levels
of myoplasmic and mitochondrial [Ca2+] are reached during
hypoxia (16, 29). These observations are all consistent
with the outcome of hypoxia/reoxygenation being determined by whether
widespread MPT has occurred. Therefore, it is critically important to
understand the interplay between Ca2+ and 
in
facilitating MPT in this setting.
In the present paper, we have demonstrated that this interplay is very
significant. Moreover, our findings suggest ways that MPT might be
avoided by appropriate manipulation of these two factors. The two
stages, to be considered separately, are MPT occurring during anoxia
and MPT occurring during reoxygenation. First, it is important to
emphasize that whereas MPT invariably causes 
dissipation, 
dissipation or Ca2+ efflux during anoxia do not necessarily
imply that MPT has occurred. As shown in Fig. 1, in isolated
mitochondria subjected to anoxia without Ca2+ loading,
complete 
dissipation and Ca2+ efflux occurred at a
critically low PO2 when electron transport was
unable to maintain the proton gradient across the inner mitochondrial membrane. 
dissipation and Ca2+ efflux under these
conditions were not due to MPT for the following reasons: 1)
mitochondrial matrix volume decreased (Fig. 3A) instead of
increasing, as occurs with MPT (Fig. 4A), 2)

dissipation and Ca2+ efflux were rapidly reversible,
recovering immediately when O2 was reintroduced (Figs. 1,
3, and 5A), which would not have been possible had MPT
occurred, 3) Ca2+ efflux during anoxia was
prevented by blocking the Ca2+ uniporter with ruthenium
red, and 4) in Ca2+-loaded mitochondria, CSA did
not prevent 
dissipation at critically low
PO2, although it abolished matrix swelling due
to MPT (Fig. 4B). A corollary to these findings is that only
mitochondrial swelling, not 
dissipation or Ca2+
efflux, was specific as an indicator of MPT under these conditions [although modest mitochondrial swelling can also result from
non-MPT-related factors such as mitochondrial ATP-sensitive K
(mitoKATP) channel activation (4)]. The
limited usefulness of 
dissipation as a criterion for MPT has
been recently discussed elsewhere (4). Finally, the
responses to anoxia/reoxygenation of isolated mitochondria under these
conditions appear identical to those of in situ mitochondria (Fig. 10).
MPT occurring during anoxia.
Whether MPT occurred during anoxia depended on the extent of matrix
Ca2+ loading, which in turn depended on 
. When
extramitochondrial [Ca2+] was increased before 
dissipation during anoxia, mitochondria accumulated Ca2+
via the Ca2+ uniporter. Depending on the respiratory
substrate and the degree of anoxia, matrix Ca2+
accumulation could first induce MPT in a subpopulation of mitochondria, producing a slow decrease 
in proportion to the size of this subpopulation (Figs. 4A and 6B), followed by MPT
in the remainder when 
dissipated at critically low
PO2 (Figs. 4A and 5B). In the latter case, the remaining mitochondria were not yet
Ca2+ loaded to a level sufficient to induce MPT at normal

, but upon 
dissipation, the Ca2+ threshold for
MPT was exceeded. Alternatively, MPT occurred in other cases only at
the time of the anoxia-induced rapid dissipation of 
(e.g., with
succinate, as in Fig. 6C). In this case, mitochondria were
insufficiently Ca2+ loaded to induce MPT at normal 
,
but upon 
dissipation, the voltage-dependent threshold of
matrix [Ca2+] required to trigger PTP opening was again
exceeded, and MPT occurred rapidly throughout the whole population of
mitochondria. This scenario is also the likely explanation for the
observation (21) that mitoKATP channel
agonists, which depolarize 
, potentiated PTP opening in normoxic
respiring isolated mitochondria subjected to Ca2+ loading.
In contrast, when extramitochondrial Ca2+ was added after

had already dissipated (Fig. 8A), Ca2+
accumulation via the Ca2+ uniporter did not occur due to
the lack of driving force for Ca2+ entry into the matrix,
and MPT was avoided during anoxia (but not necessarily after anoxia,
see MPT occurring during reoxygenation). Thus in
intact cardiac myocytes, the timing of the increase in cytoplasmic free
[Ca2+] relative to 
depolarization during anoxia is
likely to be critically important. Because mitochondrial
Ca2+ uptake accounts for approximately two-thirds of
cellular Ca2+ uptake, 
dissipation before a major
increase in extramitochondrial [Ca2+] will greatly reduce
cellular Ca2+ overload (24). In intact
myocytes, one approach toward cardioprotection is to decrease the rate
at which cytoplasmic Ca2+ increases during anoxia by
inhibiting transsarcolemmal Ca2+ influx, e.g., with
Na+/H+ exchange inhibitors (18,
31). A second approach is to prevent mitochondrial
Ca2+ uptake in polarized mitochondria, despite elevated
extramitochondrial Ca2+, by inhibiting the mitochondrial
Ca2+ uniporter, e.g., with ruthenium red, also known to be
cardioprotective (9, 28). Finally, a third approach is to
minimize mitochondrial Ca2+ accumulation by dissipating

more rapidly during anoxia, e.g., with mitoKATP
channel agonists, which is one of the mechanisms proposed to underlie
their cardioprotective effects (22, 26). The weight of
evidence now suggests that mitochondrial KATP channels are
more important than sarcolemmal KATP channels in mediating cardioprotection (17). Furthermore, direct protection of
mitochondria by mitoKATP channel agonists during
ischemia-reperfusion has recently been demonstrated
(11), consistent with prevention of MPT. These findings
make sense on general grounds because cardioprotection obviously
depends on recovery of mitochondrial function, and transient inhibition
of mitochondrial function, when induced before ischemia, could
protect them from excessive Ca2+ accumulation. The ability
of mitochondrial inhibitors, which induce full dissipation of 
,
to prevent hypercontracture and massive enzyme release, and reduce
infarct size during reoxygenation/reperfusion (12, 13, 27)
may also be an example of the former.
It is also interesting to note that even when MPT has occurred during
anoxia, as indicated by CSA-sensitive matrix swelling (e.g., Fig. 4,
A and B), we always observed at least partial
transient recovery of 
(although not matrix volume) upon
reoxygenation. This indicates that some mitochondria had still not
undergone MPT at the start of reoxygenation. Upon reoxygenation,
however, MPT was also induced in this subpopulation, as evidenced by
complete 
dissipation, and did not reverse spontaneously unless
extramitochondrial Ca2+ was removed and CSA added (see next section).
MPT occurring during reoxygenation.
Even if mitochondria have avoided accumulating sufficient
Ca2+ to induce MPT during anoxia, they are still subject to
MPT during reoxygenation (Fig. 8). In the presence of O2,
substrate, and elevated extramitochondrial [Ca2+],
partial 
recovery during reoxygenation promotes mitochondrial Ca2+ uptake via the Ca2+ uniporter. Because the
voltage dependence of Ca2+ uptake via the Ca2+
uniporter saturates at ~
110 mV (6), partial 
recovery should not appreciably limit the increase in matrix
[Ca2+]. However, the lower 
is more likely to
promote MPT at a given level of matrix Ca2+ (Figs.
5B and 8A) due to its voltage sensitivity. In
contrast, well-oxygenated and energized mitochondria can tolerate
surprisingly high Ca2+ loads without PTP opening, provided
that mitochondria are able to support normal 
. In addition,
during reoxygenation of intact cells, oxidative stress is considerably
elevated and favors PTP opening (6). Thus when
extramitochondrial Ca2+ remains high during reoxygenation,
PTP opening may be triggered even when mitochondria have not become
Ca2+ loaded during anoxia (Fig. 8). In this setting,
lowering of extramitochondrial Ca2+ is usually required to
reverse MPT, even if CSA is present (Figs. 5B,
7B, and 8A). The three approaches described above
for preventing MPT during hypoxia are also predicted to be effective at
preventing MPT during reoxygenation by limiting matrix Ca2+
accumulation: 1) reducing the extramitochondrial
[Ca2+] increase during hypoxia/reoxygenation,
2) delaying 
recovery upon reoxygenation to keep
membrane potential below the value required for Ca2+
uniporter activity, or 3) blocking Ca2+
uptake by the Ca2+ uniporter until after normal
cytoplasmic Ca2+ levels have been restored by
nonmitochondrial Ca2+ removal mechanisms.
Summary and clinical implications.
Mitochondrial 
and matrix [Ca2+] interact
strongly to regulate PTP opening during hypoxia/reoxygenation. Our
findings predict that interventions that reduce extramitochondrial
Ca2+, that prevent mitochondria from accumulating
Ca2+ by chemically inhibiting the Ca2+
uniporter, or that accelerate 
dissipation during hypoxia to limit [Ca2+] uptake via the Ca2+
uniporter are synergistic at preventing MPT during
hypoxia/reoxygenation in isolated mitochondria. Furthermore, we have
demonstrated that the fundamental responses to anoxia/reoxygenation in
isolated mitochondria under non-Ca2+-loaded conditions are
similar to those of in situ mitochondria in both permeabilized and
intact myocytes. Unfortunately, we could not determine whether
Ca2+-loaded mitochondria in situ behaved identically to
isolated mitochondria due to difficulty in controlling
extramitochondrial [Ca2+] accurately under the latter
conditions, which is a limitation of this study. Nevertheless, these
findings lend strong support to the proposition that a multipronged
strategy, combined with direct pharmacological inhibition of MPT with
CSA, will be more effective at reducing MPT during
hypoxia/reoxygenation and ischemia-reperfusion in cardiac
tissue than any of these interventions individually, thereby enhancing cardioprotection.
 |
ACKNOWLEDGEMENTS |
We thank Tan Duong for technical assistance.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant P50 HL-52319 and by the Laubisch and Kawata Endowments.
Address for reprint requests and other correspondence: J. N. Weiss, Div. of Cardiology, 3641 MRL Bldg., UCLA School of Medicine, Los Angeles, CA 90095-1760 (E-mail:
jweiss{at}mednet.ucla.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 25 August 2000; accepted in final form 18 October 2000.
 |
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