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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We studied the interplay between matrix Ca2+ concentration ([Ca2+]) and mitochondrial membrane potential (Delta psi ) in regulation of the mitochondrial permeability transition (MPT) during anoxia and reoxygenation. Without Ca2+ loading, anoxia caused near-synchronous Delta psi 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 Delta psi . However, when [Ca2+] was increased after complete Delta psi dissipation, MPT did not occur until reoxygenation, at which time reactivation of electron transport led to partial Delta psi regeneration. In the setting of elevated extramitochondrial Ca2+, this enhanced matrix Ca2+ uptake while promoting MPT because of less than full recovery of Delta psi . The interplay between Delta psi 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MYOCARDIAL VIABILITY AFTER reperfusion/reoxygenation is critically dependent on the recovery of mitochondrial membrane potential (Delta psi ) to provide the driving force for oxidative phoshorylation. A major factor that could limit recovery of Delta psi 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 Delta psi (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, Delta psi 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 Delta psi and cause PTP opening at values close to physiological Delta psi , with inhibitors doing the converse (3).

However, mitochondrial Ca2+ uptake is also regulated by Delta psi , which provides the driving force for Ca2+ uptake into the matrix through the Ca2+ uniporter. Well-maintained Delta psi promotes Ca2+ uptake when cytoplasmic free [Ca2+] increases, as occurs during ischemia and hypoxia (6). Therefore, during ischemia/hypoxia, maintenance of Delta psi 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 Delta psi 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 Delta psi 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Delta psi ) in a non-Ca2+-loaded mitochondrial suspension. A: simultaneous recording of buffer PO2 and mitochondrial Delta psi . 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 Delta psi 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 Delta psi 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.

Determination of mitochondrial matrix volume and Delta psi . 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. Delta psi was estimated by transmembrane distribution of TMRM. Changes in Delta psi 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 Delta psi 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 Delta psi 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 Delta psi with 0.5 µM FCCP. In intact cells, Delta psi 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. Delta psi recovery on reoxygenation for different groups is presented as means ± SD.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of anoxia/reoxygenation in mitochondria without Ca2+ loading. Figure 1A illustrates oxygen content and mitochondrial Delta psi in a mitochondrial suspension as a nitrogen (N2) stream was directed against buffer surface in a closed cuvette. Delta psi 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 Delta psi 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 Delta psi 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 Delta psi 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, Delta psi 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 Delta psi , irrespective of the substrates used or whether a critical level of PO2 was achieved rapidly or slowly. Figure 2 summarizes Delta psi 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 Delta psi upon reoxygenation, with different substrates, extramitochondrial Ca2+ concentration ([Ca2+]) loads, and Delta psi depolarization (DP). Delta psi 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.

Delta psi dissipation during anoxia was accompanied by slight matrix shrinkage, which reversed with Delta psi recovery upon reoxygenation (n = 3 preparations, Fig. 3A, bottom trace). Delta psi 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 Delta psi 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 Delta psi dissipation occurred during anoxia (data not shown).


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Fig. 3.   Effects of anoxia/reoxygenation on Delta psi , matrix volume (A), and extramitochondrial [Ca2+] (B) in non-Ca2+-loaded mitochondria. A: simultaneous recordings of mitochondrial Delta psi and matrix volume during anoxia (N2 arrow) and reoxygenation (O2 arrow). Matrix volume shrank with Delta psi dissipation and recovered with reoxygenation. After reoxygenation, addition of 0.5 mM EGTA had little effect on Delta psi 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 Delta psi 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 Delta psi dissipation. Upon reoxygenation, mitochondria reaccumulated Ca2+ coincident with Delta psi 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).

Effects of Ca2+-loading mitochondria before Delta psi dissipation during anoxia. When extramitochondrial Ca2+ was increased by adding small Ca2+ pulses before Delta psi dissipation during anoxia, a different response was observed. With pyruvate as the substrate, only a few 5-µM Ca2+ pulses were required to accelerate Delta psi dissipation in a subpopulation of mitochondria before PO2 reached the critical level inhibiting electron transport, whereupon Delta psi 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 Delta psi dissipation (n = 6 preparations, Fig. 4A). Upon reoxygenation, there was partial, transient recovery of Delta psi , followed by Delta psi 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, Delta psi 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 Delta psi dissipation during anoxia: effects on Delta psi 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 Delta psi dissipation caused initially slow Delta psi depolarization and matrix swelling consistent with mitochondrial permeability transition (MPT) in a subpopulation of the mitochondria, followed by near-synchronous rapid Delta psi dissipation and further matrix swelling in the remainder. Reoxygenation (O2 arrow) led to transient partial Delta psi recovery, followed by Delta psi dissipation and further matrix swelling. Addition of both EGTA and succinate were required for recovery of Delta psi 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 Delta psi depolarization or matrix swelling, although near-synchronous Delta psi dissipation still occurred at critically low PO2. Changes reversed rapidly with reoxygenation alone, indicating that MPT had been prevented (confirmed in 3 preparations).

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 Delta psi dissipated abruptly during anoxia, favoring MPT by way of inner membrane depolarization. The increase in extramitochondrial [Ca2+] during anoxia decreased Delta psi recovery on reoxygenation in succinate-energized mitochondria (n = 4 preparations, Fig. 2). Finally, with more gradual onset of Delta psi dissipation during partial anoxia, a similar pattern of slow partial Delta psi 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 Delta psi in kidney cells during hypoxia/reoxygenation (32).


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Fig. 5.   Ca2+-loading mitochondria before Delta psi 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 Delta psi 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 Delta psi dissipation or matrix swelling indicating MPT. When critically low PO2 caused rapid Delta psi 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 Delta psi 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 Delta psi 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 Delta psi and reversal of matrix swelling (3 preparations). A-C: mitochondria (0.3 mg/ml) were energized with 5 mM succinate, and Delta psi was recorded with TMRM.

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 Delta psi 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 Delta psi 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 Delta psi 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 Delta psi had recovered fully. As shown in Figs. 4A and 5B, Delta psi 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 Delta psi dissipation. However, CSA was often unable to restore Delta psi 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 Delta psi 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 Delta psi dissipation that reversed spontaneously upon reoxygenation and was reproduced by Delta psi 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 Delta psi dissipation. On reoxygenation, mitochondria initially reaccumulated Ca2+ coincident with Delta psi 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 Delta psi 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.

Effects of Ca2+ loading after Delta psi dissipation during anoxia. In the previous experiments, extramitochondrial Ca2+ was added before Delta psi dissipated during anoxia. During anoxia in intact cardiac myocytes, however, Ca2+ may continue to rise after Delta psi collapses (8), generating a possible situation in which rapid Delta psi recovery during reoxygenation could result in significant Ca2+ uptake and trigger MPT. The resulting Delta psi 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 Delta psi 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, Delta psi began to recover. However, the recovery was transient and followed by rapid depolarization and matrix swelling, indicating Ca2+-dependent MPT (n = 5 preparations). Both Delta psi and matrix volume recovered after addition of EGTA and CSA, confirming that changes were Ca2+ dependent and CSA sensitive (n = 5 preparations). Thus Delta psi 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 Delta psi dissipation during anoxia. A: two 25 µM Ca2+ pulses administered after Delta psi dissipation during anoxia (N2) had no further effect on Delta psi or matrix volume. Upon reoxygenation, Delta psi recovery was partial and transient, followed by Delta psi 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 Delta psi 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 Delta psi recorded with Calcium Green-5N and TMRM, respectively.

Figure 8B shows that no Ca2+ uptake occurred when 10-µM Ca2+ pulses were added after Delta psi 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 Delta psi regeneration in the presence of high extramitochondrial [Ca2+] could also be demonstrated by dissipating Delta psi 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 Delta psi 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 Delta psi 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 Delta psi (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).

Effect of anoxia/reoxygenation on mitochondrial Delta psi 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 Delta psi (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, Delta psi rapidly and synchronously dissipated. Turning off the N2 stream led to slow recovery of Delta psi that dissipated rapidly when N2 was turned back on. Subsequent reoxygenation with an O2 stream resulted in immediate recovery of Delta psi (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), Delta psi (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 Delta psi 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), Delta psi (top trace) rapidly decreased, but incompletely. Subsequent addition of 10 µM oligomycin (Oligo) caused additional Delta psi 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. Delta psi 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 Delta psi until PO2 reached a critical level, at which point Delta psi dissipated rapidly and synchronously, but to an incomplete extent. The partial Delta psi 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 Delta psi (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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta psi are two of the most crucial. Elevated mitochondrial matrix [Ca2+] is regarded as the most essential requirement, and Delta psi 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 Delta psi (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 Delta psi 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 Delta psi dissipation, Delta psi 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 Delta psi 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. Delta psi 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) Delta psi 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 Delta psi 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 Delta psi 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 Delta psi 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 Delta psi . When extramitochondrial [Ca2+] was increased before Delta psi 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 Delta psi in proportion to the size of this subpopulation (Figs. 4A and 6B), followed by MPT in the remainder when Delta psi 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 Delta psi , but upon Delta psi 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 Delta psi (e.g., with succinate, as in Fig. 6C). In this case, mitochondria were insufficiently Ca2+ loaded to induce MPT at normal Delta psi , but upon Delta psi 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 Delta psi , potentiated PTP opening in normoxic respiring isolated mitochondria subjected to Ca2+ loading.

In contrast, when extramitochondrial Ca2+ was added after Delta psi 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 Delta psi depolarization during anoxia is likely to be critically important. Because mitochondrial Ca2+ uptake accounts for approximately two-thirds of cellular Ca2+ uptake, Delta psi 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 Delta psi 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 Delta psi , 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 Delta psi (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 Delta psi 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 Delta psi 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 Delta psi recovery should not appreciably limit the increase in matrix [Ca2+]. However, the lower Delta psi 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 Delta psi . 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 Delta psi 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 Delta psi 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 Delta psi 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Altschuld, RA, Wenger WC, Lamka KG, Kindig OR, Capen CC, Mizuhira V, Vander Heide RS, and Brierley GP. Structural and functional properties of adult rat heart myocytes lysed with digitonin. J Biol Chem 260: 14325-14334, 1985[Abstract/Free Full Text].

2.   Bernardi, P. Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol Rev 79: 1127-1155, 1999[Abstract/Free Full Text].

3.   Bernardi, P. The permeability transition pore: control points of a cyclosporin A-sensitive mitochondrial channel involved in cell death. Biochim Biophys Acta 1275: 5-9, 1996[ISI][Medline].

4.   Bernardi, P, Scorrano L, Colonna R, Petronilli V, and Di Lisa F. Mitochondria and cell death. Mechanistic aspects and methodological issues. Eur J Biochem 264: 687-701, 1999[Abstract/Free Full Text].

5.   Bernardi, P, Veronese P, and Petronilli V. Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore. I. Evidence for two separate Me2+ binding sites with opposing effects on the pore open probability. J Biol Chem 268: 1005-1010, 1993[Abstract/Free Full Text].

6.   Crompton, M. The mitochondrial permeability transition pore and its role in cell death. Biochem J 341: 233-249, 1999[ISI][Medline].

7.   Delcamp, TJ, Dales C, Ralenkotter L, Cole PS, and Hadley RW. Intramitochondrial [Ca2+] and membrane potential in ventricular myocytes exposed to anoxia-reoxygenation. Am J Physiol Heart Circ Physiol 275: H484-H494, 1998[Abstract/Free Full Text].

8.   Di Lisa, F, Blank PS, Colonna R, Gambassi G, Silverman HS, Stern MD, and Hansford RG. Mitochondrial membrane potential in single living adult rat cardiac myocytes exposed to anoxia or metabolic inhibition. J Physiol 486: 1-13, 1995[Abstract].

9.   Ferrari, R. The role of mitochondria in ischemic heart disease. J Cardiovasc Pharmacol 28, Suppl1: S1-10, 1996[ISI][Medline].

10.   Fontaine, E, Eriksson O, Ichas F, and Bernardi P. Regulation of the permeability transition pore in skeletal muscle mitochondria. Modulation by electron flow through the respiratory chain complex I. J Biol Chem 273: 12662-12668, 1998[Abstract/Free Full Text].

11.   Fryer, RM, Eells JT, Hsu AK, Henry MM, and Gross GJ. Ischemic preconditioning in rats: role of mitochondrial K(ATP) channel in preservation of mitochondrial function. Am J Physiol Heart Circ Physiol 278: H305-H312, 2000[Abstract/Free Full Text].

12.   Ganote, CE, McGarr J, Liu SY, and Kaltenbach JP. Oxygen-induced enzyme release. Assessment of mitochondrial function in anoxic myocardial injury and effects of the mitochondrial uncoupling agent 2,4-dinitrophenol (DNP). J Mol Cell Cardiol 12: 387-408, 1980[ISI][Medline].

13.   Ganote, CE, Worstell J, and Kaltenbach JP. Oxygen-induced enzyme release after irreversible myocardial injury. Effects of cyanide in perfused rat hearts. Am J Pathol 84: 327-350, 1976[Abstract].

14.   Goldhaber, JI, and Liu E. Excitation-contraction coupling in single guinea-pig ventricular myocytes exposed to hydrogen peroxide. J Physiol 477: 135-147, 1994[Abstract].

15.   Griffiths, EJ, and Halestrap AP. Protection by cyclosporin A of ischemia-reperfusion-induced damage in isolated rat hearts. J Mol Cell Cardiol 25: 1461-1469, 1993[ISI][Medline].

16.   Griffiths, EJ, Ocampo CU, Savage JS, Rutter GA, Hansford RG, Stern MD, and Silverman HS. Mitochondrial calcium transporting pathways during hypoxia and reoxygenation in single rat cardiomyocytes. Cardiovasc Res 39: 423-433, 1998[ISI][Medline].

17.   Grover, GJ, and Garlid KD. ATP-sensitive potassium channels: a review of their cardioprotective pharmacology. J Mol Cell Cardiol 32: 677-695, 2000[ISI][Medline].

18.  Gumina RJ, Buerger E, Eickmeier C, Moore J, Daemmgen J, and Gross GJ. Inhibition of the Na+/H+ exchanger confers greater cardioprotection against 90 min of myocardial ischemia than ischemic preconditioning in dogs. Circulation 100: 2519-2526; discussion 2469-2472, 1999.

19.   Gunter, TE, Gunter KK, Sheu SS, and Gavin CE. Mitochondrial calcium transport: physiological and pathological relevance. Am J Physiol Cell Physiol 267: C313-C339, 1994[Abstract/Free Full Text].

20.   Haworth, RA, and Hunter DR. Allosteric inhibition of the Ca2+-activated hydrophilic channel of the mitochondrial inner membrane by nucleotides. J Membr Biol 54: 231-236, 1980[ISI][Medline].

21.   Holmuhamedov, EL, Jovanovic S, Dzeja PP, Jovanovic A, and Terzic A. Mitochondrial ATP-sensitive K+ channels modulate cardiac mitochondrial function. Am J Physiol Heart Circ Physiol 275: H1567-H1576, 1998[Abstract/Free Full Text].

22.   Holmuhamedov, EL, Wang L, and Terzic A. ATP-sensitive K+ channel openers prevent Ca2+ overload in rat cardiac mitochondria. J Physiol 519: 347-360, 1999[Abstract/Free Full Text].

23.   Ichas, F, Jouaville LS, and Mazat JP. Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals. Cell 89: 1145-1153, 1997[ISI][Medline].

24.   Korge, P, and Langer GA. Mitochondrial Ca2+ uptake, efflux, and sarcolemmal damage in Ca2+-overloaded cultured rat cardiomyocytes. Am J Physiol Heart Circ Physiol 274: H2085-H2093, 1998[Abstract/Free Full Text].

25.   Korge, P, and Weiss JN. Thapsigargin directly induces the mitochondrial permeability transition. Eur J Biochem 265: 273-280, 1999[Abstract/Free Full Text].

26.   Liu, Y, Sato T, O'Rourke B, and Marban E. Mitochondrial ATP-dependent potassium channels: novel effectors of cardioprotection? Circulation 97: 2463-2469, 1998[Abstract/Free Full Text].

27.   Minners, J, Van den Bos EJ, Yellon DM, Schwalb H, Opie LH, and Sack MN. Dinitrophenol, cyclosporin A, and trimetazidine modulate preconditioning in the isolated rat heart: support for mitochondrial role in cardioprotection. Cardiovasc Res 47: 68-73, 2000[ISI][Medline].

28.   Miyamae, M, Camacho SA, Weiner MW, and Figueredo VM. Attenuation of postischemic reperfusion injury is related to prevention of [Ca2+]m overload in rat hearts. Am J Physiol Heart Circ Physiol 271: H2145-H2153, 1996[Abstract/Free Full Text].

29.   Miyata, H, Lakatta EG, Stern MD, and Silverman HS. Relation of mitochondrial and cytosolic free calcium to cardiac myocyte recovery after exposure to anoxia. Circ Res 71: 605-613, 1992[Abstract].

30.   Nazareth, W, Yafei N, and Crompton M. Inhibition of anoxia-induced injury in heart myocytes by cyclosporin A. J Mol Cell Cardiol 23: 1351-1354, 1991[ISI][Medline].

31.   Tani, M, and Neely JR. Role of intracellular Na+ in Ca2+ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Possible involvement of H+-Na+ and Na+-Ca2+ exchange. Circ Res 65: 1045-1056, 1989[Abstract].

32.   Weinberg, JM, Venkatachalam MA, Roeser NF, and Nissim I. Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates. Proc Natl Acad Sci USA 97: 2826-2831, 2000[Abstract/Free Full Text].


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