Although no doubt exists that the maintenance of tissue viability requires optimal mitochondrial function, the ensuing cell death is often determined by mitochondria. This concept is made dramatically clear by the sequence of events that characterizes the ischaemic damage in the heart. Three major phases can be described (Di Lisa et al. 1998). The first is associated with the onset of ischaemia, and changes mitochondria from ATP producers into powerful ATP utilizers (Di Lisa et al. 1995). During this phase, the inverse operation of F₀F₁ ATPase maintains the mitochondrial membrane potential by using the ATP made available by glycolysis. The second phase can be identified from the functional and structural alterations of mitochondria caused by prolongation of ischaemia, such as decreased utilization of NAD-linked substrates, release of cytochrome c and involvement of mitochondrial channels. These events indicate that the relationship between ischaemic damage and mitochondria is not limited to the failure in ATP production. Finally, the third phase links mitochondria to the destiny of the myocytes upon post-ischaemic reperfusion. Indeed, depending on the duration and the severity of ischaemia, not only is mitochondrial function necessary for cell recovery, but it can also exacerbate cell injury. Indeed, it is generally accepted that the rupture of sarcolemma results from an uncontrolled activation of contraction in cells lacking the possibility of relaxation. Such a condition results from a suboptimal recovery of mitochondrial function because reduced contents of ATP co-exist with elevated concentrations of intracellular Ca²⁺.

A relevant link between intracellular Ca²⁺ overload and mitochondrial dysfunction is represented by the opening of the mitochondrial permeability transition pore (Bernardi, 1999). The permeability transition is a regulated permeability increase of the mitochondrial inner membrane to solutes with molecular masses up to 1500 Da mediated by opening of a high-conductance channel, the permeability transition pore (PTP), whose molecular nature remains debated. The PTP is modulated by a variety of effectors of cell death, including calcium, lipid mediators and reactive oxygen species (Bernardi, 1999). We have recently elucidated the role of PTP in the reperfusion damage by investigating NAD⁺ metabolism (Di Lisa et al. 2001; Di Lisa & Ziegler, 2001). In fact, mitochondrial NAD⁺ content, which is hardly affected during ischaemia, becomes almost depleted when coronary flow is restored after a prolonged period of ischaemia. The inhibition of mitochondrial NAD⁺ depletion exerted by CsA suggests that upon reperfusion the rise in intracellular Ca²⁺, along with the recovery of neutral pH and the boosting of oxyradical generation, promotes PTP opening, causing the release of intramitochondrial NAD⁺ and its subsequent hydrolysis. Not only is the decrease of mitochondrial NAD⁺ prevented when PTP is inhibited, but also tissue viability is significantly protected (Di Lisa et al. 2001). Besides affecting energy metabolism, the mitochondrial release of NAD⁺ is likely to modify several intracellular processes triggered by ischaemia or other pathological conditions. Indeed, once released out of the mitochondrial matrix, NAD⁺ could be transformed into cyclic ADP ribose which promoting Ca²⁺ release from intracellular stores may amplify and extend the effects exerted by an initial rise in intracellular [Ca²⁺]. Thus the release of NAD⁺ from mitochondria and its subsequent utilization within other cell compartments could be part of the mechanisms through which mitochondria transduce and amplify an initial trigger provided by reperfusion (Di Lisa & Ziegler, 2001).

Besides the involvement in necrosis, alterations of mitochondrial structure and function appear pivotal in the commitment of cells to apoptosis. Indeed, a severe reduction of the mitochondrial membrane potential, the opening of the permeability transition pore and/or the release of pro-apoptotic proteins often precede the appearance of other characteristic signs of apoptosis, such as phosphatidylserine or DNA fragmentation (Bernardi et al. 1999; Hengartner, 2000). The causal relationships between these processes are still debated (Hengartner, 2000; Bernardi et al. 2001). A key issue is to assess whether, and when, PTP opening occurs in the course of apoptosis and what are its consequences in situ. We investigated the relationship between PTP opening, mitochondrial depolarization, cytochrome c release and occurrence of cell death (Petronilli et al. 2001). Using a technique developed in our laboratory (Petronilli et al. 1999), we could detect both transient and long-lasting PTP openings. While cell viability is hardly affected by PTP openings of short duration, longer PTP openings cause mitochondrial depolarization followed by release of cytochrome c and apoptosis. Thus modulation of PTP open time appears to be the key element in determining the outcome of stimuli that converge on the PTP.

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