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Cardiovascular Research 1999 44(3):470-473; doi:10.1016/S0008-6363(99)00368-5
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

The mitochondrial permeability transition and the calcium, oxygen and pH paradoxes: one paradox after another

John J Lemasters*

Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Room 236, Taylor Hall, Chapel Hill, NC 27599-7090, USA

* Tel.: +1-919-966-5507; fax: +1-919-966-1856 lemaster{at}med.unc.edu

accepted 13 October 1999

See article by Ban et al. [3] (pages 556–567) in this issue.

In the calcium paradox, Ca2+-free incubation produces Na+ loading of cardiac myocytes largely mediated by Na+ influx via L-type Ca2+ channels. Subsequent exposure to normal extracellular Ca2+ activates sarcolemmal Na+/Ca2+ exchange operating in the reverse mode. As a consequence, cytosolic free Ca2+ rapidly increases causing an abrupt and apparently irreversible contracture of the myocytes [1]. The calcium paradox has many parallels to the oxygen paradox of anoxia/reoxygenation and ischemia/reperfusion injuries where Na+ loading occurs during anoxia/ischemia, which is followed by Ca2+ overload and irreversible contracture after reoxygenation/reperfusion [2].

The interesting and provocative paper by Ban et al. in this issue of Cardiovascular Research [3] addresses mitochondrial changes occurring during the calcium paradox. After Ca2+ repletion following a Ca2+-free incubation, mitochondria of isolated guinea pig cardiac myocytes depolarized completely after a delay of 60–90 s. This event was accompanied by oxidation of mitochondrial NAD(P)H and hydrolysis of ATP, the latter measured indirectly by an increase of free Mg2+. Mg2+ forms a chelate with ATP but not ADP or AMP and thus free Mg2+ increases and decreases as ATP hydrolysis and resynthesis occur in models of anoxia/reoxygenation [4,5]. Taken together, the findings of Ban et al. [3] signify a massive uncoupling of oxidative phosphorylation. Such uncoupling represents a devastating metabolic disruption since uncoupled mitochondria not only fail to synthesize ATP but also actively hydrolyze ATP. This cellular state of affairs is worse than having no mitochondria at all [6].

Why does mitochondrial uncoupling occur? Ban et al. [3] address two hypotheses. The first is that Ca2+ overload causes futile energy-dissipating mitochondrial Ca2+ cycling. This cycling involves mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter driven by the mitochondrial membrane potential ({Delta}{psi}m) and mitochondrial Ca2+ release by the mitochondrial Na2+/Ca2+ exchanger. Since mitochondrial Na+/Ca2+ exchange catalyzes the electrogenic interchange of three Na+ for one Ca2+ [7], both the uptake and release of Ca2+ dissipate {Delta}{psi}m. Support for Ca2+ cycling as the basis for mitochondrial depolarization in the calcium paradox comes from observations by Ban et al. [3] that ruthenium red, an inhibitor of the electrogenic mitochondrial Ca2+ uniporter [8], and CGP-37157, an inhibitor of mitochondria Na+/Ca2+ exchange [9], both block mitochondrial depolarization, NAD(P)H oxidation, and cellular ATP depletion after Ca2+ repletion. Thus, blockade of either mitochondrial Ca2+ uptake or release is sufficient to prevent mitochondrial depolarization.

An alternate hypothesis is that mitochondrial Ca2+ overloading causes onset of the mitochondrial permeability transition (MPT). The MPT occurs when a very high conductance permeability transition (PT) pore opens in the mitochondrial inner membrane (reviewed in Ref. [10]). This pore conducts solutes of molecular weight up to 1500 Da. Increased Ca2+ and generation of reactive oxygen species promote PT pore opening, which in turn causes mitochondrial depolarization, large amplitude mitochondrial swelling and uncoupling of oxidative phosphorylation. Cyclosporin A at nanomolar concentrations inhibits the MPT. Ban et al. [3] found that cyclosporin A at a concentration of only 200 nM blocked mitochondrial depolarization, NAD(P)H oxidation and cellular ATP depletion after Ca2+ repletion, implicating that PT pore opening is the basis for mitochondrial uncoupling in the calcium paradox. A role for PT pore opening in the calcium paradox, however, would seem to be contradicted by the effect of CGP-37157. By blocking Ca2+ efflux through the mitochondrial Na+/Ca2+ exchanger, CGP-37157 should increase mitochondrial Ca2+ and promote the MPT and mitochondrial depolarization. Instead, CGP-37157 blocked mitochondrial depolarization.

To explain this discrepancy, Ban et al. [3] suggested that cyclosporin A blocks mitochondrial Na+/Ca2+ exchange, but virtually no data in the literature supports this action of cyclosporin A at the nanomolar concentrations used in their study. Other explanations are possible. For example, mitochondrial Na+/Ca2+ exchange acting in the reverse mode may promote mitochondrial Ca2+ uptake, as proposed for mitochondria in anoxic myocytes [11]. This mitochondrial Ca2+ may then, in turn, induce onset of the MPT. Indeed, confocal microscopy of ATP-depleted myocytes shows a sequence of increased cytosolic Ca2+ and then increased mitochondrial Ca2+, which is followed by mitochondrial depolarization and subsequent Ca2+ release [12]. These last changes suggest onset of the MPT. More direct evidence comes from experiments showing that cyclosporin A prevents lethal cell injury in a variety of models of anoxia/reoxygenation, ischemia/reperfusion, excitotoxicity and oxidative stress to cardiac myocytes, hepatocytes, endothelial cells, neurons and other cells (reviewed in Ref. [13]). Moreover, onset of cell death in these models is associated with a cyclosporin A-sensitive increase of non-specific permeability of the mitochondrial inner membrane, as determined with radioactive tracers or imaged directly by confocal microscopy [14–16].

Mitochondrial Na+/Ca2+ exchange is coupled secondarily to mitochondrial H+/Na+ exchange to produce net H+/Ca2+ exchange. When cytosolic Ca2+ is high, exchange of cytosolic Ca2+ for intramitochondrial H+ can act to alkalinize the matrix. PT pore conductance depends on a high matrix pH. Thus, inhibition of Na+/Ca2+ exchange by CGP-37157 may prevent matrix alkalization and thereby inhibit or delay onset of the MPT. This postulated mechanism could account for the protection by CGP-37157 against mitochondrial depolarization in the calcium paradox, but experimental data are needed to prove such a hypothesis. pH is very important in ischemia/reperfusion injury, a fact that is perhaps still under appreciated. During ischemia, tissue pH drops by a unit or more. This naturally occurring acidosis strongly protects against the onset of necrotic cell death in virtually all cell types studied [17–19]. When normal pH recovers after reperfusion, however, cell killing accelerates dramatically [19–23]. This pH-dependent reperfusion injury, or pH paradox, occurs as acidotic intracellular pH recovers to near normal ({approx}pH 7) after reperfusion [21,22]. Moreover, the pH paradox is independent of whether the cells or tissues are actually reoxygenated.

A pH-dependent MPT underlies the pH paradox. During reperfusion, nonspecific mitochondrial membrane permeability abruptly increases as acidotic intracellular pH recovers towards normal. Reperfusion at acidotic pH or with cyclosporin A blocks this increase of mitochondrial membrane permeability, which allows mitochondrial repolarization to occur and prevents cell death [24].

The MPT is also implicated in Ca2+ overload injury caused by the Ca2+ ionophore, Br-A23187. In hepatocytes, Br-A23187 produces an increase of cytosolic and mitochondrial free Ca2+, which is followed by an increase of nonspecific mitochondrial membrane permeability. This increase of permeability can be visualized directly by confocal microscopy from the redistribution of the 620 Da fluorophore, calcein, from the cytosol into the mitochondria. Cell death then follows this increase of mitochondrial membrane permeability. Cyclosporin A prevents both cell death and the increase of mitochondrial membrane permeability after exposure to Ca2+ ionophore but not the increase of cytosolic and mitochondrial free Ca2+ [25]. Thus, it is plausible that the MPT also plays a crucial role in mitochondrial depolarization and uncoupling after Ca2+ overloading of myocytes in the calcium paradox.

Although classically associated with necrotic cell death, ischemia and hypoxia also cause apoptosis in cells from heart and other tissues [26]. Similarly, the MPT can play a key role in signaling apoptosis [27], as confirmed directly by confocal imaging [28]. Large amplitude swelling after onset of the MPT ruptures the mitochondrial outer membrane and releases cytochrome c and other pro-apoptotic factors. Cytochrome c then promotes activation of a cascade of cytosolic cysteine-asparate proteases called caspases, particularly caspases 9 and 3. Through interaction with cytochrome c, APAF-1 (apoptosis activating factor 1) and ATP (or dATP), caspase 9 converts pro-caspase 3 to caspase 3, the principal ‘executioner’ caspase whose activity leads directly to the outward manifestations of apoptosis, such as internucleosomal DNA fragmentation [29,30].

Although we should not assume that the MPT causes all forms of apoptotic and necrotic cell death, the MPT is in the peculiar position of initiating both apoptosis and necrosis. A deciding factor in whether apoptotic or necrotic cell death ensues after the MPT may be cellular ATP levels. If ATP is profoundly depleted, then cytochrome c cannot activate caspase 3 through the APAF-1/caspase 9 pathway [29]. Instead, cells must die a necrotic death, which occurs rapidly in highly aerobic, ATP-consuming cells like myocytes. However, if ATP levels are maintained above 10–20% of normal by glycolysis or from ATP synthesis by a subpopulation of intact mitochondria not ongoing the MPT, then necrotic cell death is averted and apoptosis progresses instead [25]. Thus, ATP acts as a switch between apoptosis and necrosis [31–33], which is operated by dynamic balance between ATP generation by glycolysis and still intact mitochondria and ATP consumption by uncoupled mitochondria and other ATP-consuming cellular processes [25]. In this way, the MPT is a shared pathway leading to both apoptosis and necrosis. Indeed, after ischemia/reperfusion and other tissue stresses, cellular features of both apoptosis and necrosis often co-exist. Pure apoptosis and pure necrotic cell death thus represent extremes in a continuous spectrum of cellular changes in dying cells. Recently, the term ‘necrapoptosis’ was introduced to emphasize that apoptosis and necrosis share common pathways and may not be so distinct as first proposed [34].

Future research will be needed to determine whether or not the MPT actually mediates mitochondrial depolarization and cellular ATP depletion in the pH paradox, but methods such as laser scanning confocal microscopy are now available to evaluate mitochondrial membrane permeability in single mitochondria of individual cardiac myocytes as injury progresses. The MPT has other possible implications for heart disease. In cardiac failure, individual myocytes are stimulated to work harder, an effect mediated in part by adrenergic signals and an increase of in average cytosolic and mitochondrial free Ca2+. Increased mitochondrial Ca2+ and increased cellular ATP demand together stimulate mitochondrial respiration and the associated production of reactive oxygen species that occurs as a byproduct of mitochondrial respiration [35]. Increased Ca2+ and free radical generation predispose to the MPT. Then as the MPT occurs in single mitochondria, the remaining mitochondria must respire even faster to keep pace with energy demand. Free radical production rises further, cytosolic Ca2+ increases more, and a vicious cycle may ensue of increasing mitochondrial dysfunction through onset of the MPT. This culminates in apoptotic cell death. As cell number declines, the factors driving the MPT and apoptosis may amplify to cause progressive heart failure. This is a hypothetical scenario, to be sure, but concrete support is beginning to emerge, for example, from experimental evidence showing a key role of the MPT in doxorubicin-induced cardiotoxicity [36].

As we go from one paradox to the next, we do in fact make progress. The discovery in the mitochondrial permeability transition of a potential common mechanism for the calcium, oxygen and pH paradoxes carries the implication that powerful and broad-acting new therapeutic strategies may one day be developed. Development of selective blockers of the PT pore represents an important and novel target for drug discovery. This brings us to yet another paradox: the more we know, the more we need to learn.


   Acknowledgements
 
The author is supported, in part, by Grants AA09156, AG07218, AG13637, DK37034 and HL27430 from the National Institutes of Health.


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