The occurrence and the mode of opening of the mitochondrial permeability transition pore (MTP) were investigated directly in intact cells by monitoring the fluorescence of mitochondrial entrapped calcein. When MH1C1 cells and hepatocytes were loaded with calcein AM, calcein was also present within mitochondria, because (i) its mitochondrial signal was quenched by the addition of tetramethylrhodamine methyl ester and (ii) calcein-loaded mitochondria could be visualized after digitonin permeabilization. Under the latter condition, the addition of Ca2+ induced a prompt and massive release of the accumulated calcein, which was prevented by CsA, indicating that calcein release could, in principle, probe MTP opening in intact cells as well. To study this process, we developed a procedure by which the cytosolic calcein signal was quenched by Co2+. In hepatocytes and MH1C1 cells coloaded with Co2+ and calcein AM, treatment with MTP inducers caused a rapid, though limited, decrease in mitochondrial calcein fluorescence, which was significantly reduced by CsA. We also observed a constant and spontaneous decrease in mitochondrial calcein fluorescence, which was completely prevented by CsA. Thus MTP likely fluctuates rapidly between open and closed states in intact cells.

Mitochondria are involved in cell death for reasons that go beyond ATP supply. A recent advance has been the discovery that mitochondria contain and release proteins that are involved in the apoptotic cascade, like cytochrome c and apoptosis inducing factor. The involvement of mitochondria in cell death, and its being cause or consequence, remain issues that are extremely complex to address in situ. The response of mitochondria may critically depend on the type of stimulus, on its intensity, and on the specific mitochondrial function that has been primarily perturbed. On the other hand, the outcome also depends on the integration of mitochondrial responses that cannot be dissected easily. Here, we try to identify the mechanistic aspects of mitochondrial involvement in cell death as can be derived from our current understanding of mitochondrial physiology, with special emphasis on the permeability transition and its consequences (like onset of swelling, cytochrome c release and respiratory inhibition); and to critically evaluate methods that are widely used to monitor mitochondrial function in situ.Keywords: mitochondria; cell death; apoptosis; necrosis; permeability transition; calcium; caspases; fluorescence; probes; cytochrome c.Mitochondria are central to the life of eukaryotic cells. In recent years, it has become clear that mitochondria also play a key role in the pathways to cell death [1±8]. This role of mitochondria cannot be explained by a mere`loss of function' resulting in an energetic deficit. Rather, it is increasingly recognized as aǹ active' process mediated by regulated effector mechanisms in a wide variety of conditions. A large body of observations from the fields of immunology, toxicology, oncology, neurology and cardiology testifies about the centrality of this issue. However, the general reader is probably puzzled by the growing number of conflicting reports, and by the diverging conclusions reached by different groups when mechanistic issues are addressed and/ or cause-relationship effects are discussed. A clear example is represented by the question of cytochrome c release, and of how this event relates to mitochondrial respiration, maintenance of the mitochondrial membrane potential difference (Dc m ) and volume homeostasis.In our opinion, part of the problem resides in the intrinsic difficulties one faces when measuring`mitochondrial function' in intact cells, an issue that is not always adequately appreciated. Mitochondrial responses in vivo may be extremely hard to decipher, and a large discrepancy currently exists between our understanding of mitochondrial function in the isolated organelle and in the intact cell. However, an acceptable description of in vivo events must always take into account facts and concepts established in decades of in vitro studies. Also, it should be realized that discrepancies may arise from inapparent differences in the experimental conditions, but also from artifacts or misinterpretation of the in situ experiments. As should become clear later in the revie...

The mitochondrial permeability transition pore is a high conductance channel whose opening leads to an increase of mitochondrial inner membrane permeability to solutes with molecular masses up to ≈ 1500 Da. In this review we trace the rise of the permeability transition pore from the status of in vitro artifact to that of effector mechanism of cell death. We then cover recent results based on genetic inactivation of putative permeability transition pore components, and discuss their meaning for our understanding of pore structure. Finally, we discuss evidence indicating that the permeability transition pore plays a role in pathophysiology, with specific emphasis on in vivo models of disease.

The opening of the mitochondrial permeability transition pore (PTP) has been suggested to play a key role in various forms of cell death, but direct evidence in intact tissues is still lacking. We found that in the rat heart, 92% of NAD ؉ glycohydrolase activity is associated with mitochondria. This activity was not modified by the addition of Triton X-100, although it was abolished by mild treatment with the protease Nagarse, a condition that did not affect the energy-linked properties of mitochondria. The addition of Ca 2؉ to isolated rat heart mitochondria resulted in a profound decrease in their NAD ؉ content, which followed mitochondrial swelling. Cyclosporin A(CsA), a PTP inhibitor, completely prevented NAD ؉ depletion but had no effect on the glycohydrolase activity. Thus, in isolated mitochondria PTP opening makes NAD ؉ available for its enzymatic hydrolysis. Perfused rat hearts subjected to global ischemia for 30 min displayed a 30% decrease in tissue NAD ؉ content, which was not modified by extending the duration of ischemia. Reperfusion resulted in a more severe reduction of both total and mitochondrial contents of NAD ؉ , which could be measured in the coronary effluent together with lactate dehydrogenase. The addition of 0.2 M CsA or of its analogue MeVal-4-Cs (which does not inhibit calcineurin) maintained higher NAD ؉ contents, especially in mitochondria, and significantly protected the heart from reperfusion damage, as shown by the reduction in lactate dehydrogenase release. Thus, upon reperfusion after prolonged ischemia, PTP opening in the heart can be documented as a CsA-sensitive release of NAD ؉ , which is then partly degraded by glycohydrolase and partly released when sarcolemmal integrity is compromised. These results demonstrate that PTP opening is a causative event in reperfusion damage of the heart. Depending on the duration and severity of myocardial ischemia, reperfusion can result in either recovery of contractile function or rapid transition toward tissue necrosis (for review see Refs. 1-3). Paradoxically, both events require coupled mitochondrial respiration (4). Indeed, cyanide (5) or 2,4-dinitrophenol (6) largely reduce the release of intracellular enzymes, the marker of cell death induced by postischemic reperfusion. However, after more than 25 years, the specific mechanisms underlying these phenomenological observations have yet to be elucidated.A large body of experimental evidence suggests that a suboptimal mitochondrial function could produce low levels of ATP, which in the presence of even a modest rise in [Ca 2ϩ ] i might cause hypercontracture in isolated cardiomyocytes (7) and sarcolemma rupture in intact hearts (8,9). Such a sequence of events could be set in motion by the opening of the mitochondrial PTP, 1 a high conductance channel located in the inner mitochondrial membrane (10). The open probability of this channel is regulated by several factors including mitochondrial membrane potential difference (⌬ m ), Ca 2ϩ , matrix pH, and CsA, a high affinity inhibitor (1...

SummaryMitochondrial morphological and ultrastructural changes occur during apoptosis and autophagy, but whether they are relevant in vivo for tissue response to damage is unclear. Here we investigate the role of the optic atrophy 1 (OPA1)-dependent cristae remodeling pathway in vivo and provide evidence that it regulates the response of multiple tissues to apoptotic, necrotic, and atrophic stimuli. Genetic inhibition of the cristae remodeling pathway in vivo does not affect development, but protects mice from denervation-induced muscular atrophy, ischemic heart and brain damage, as well as hepatocellular apoptosis. Mechanistically, OPA1-dependent mitochondrial cristae stabilization increases mitochondrial respiratory efficiency and blunts mitochondrial dysfunction, cytochrome c release, and reactive oxygen species production. Our results indicate that the OPA1-dependent cristae remodeling pathway is a fundamental, targetable determinant of tissue damage in vivo.