Reperfusion of the heart after a period of ischaemia leads to the opening of a nonspecific pore in the inner mitochondrial membrane, known as the mitochondrial permeability transition pore (MPTP). This transition causes mitochondria to become uncoupled and capable of hydrolysing rather than synthesising ATP. Unrestrained, this will lead to the loss of ionic homeostasis and ultimately necrotic cell death. The functional recovery of the Langendorff-perfused heart from ischaemia inversely correlates with the extent of pore opening, and inhibition of the MPTP provides protection against reperfusion injury. This may be mediated either by a direct interaction with the MPTP [e.g., by Cyclosporin A (CsA) and Sanglifehrin A (SfA)], or indirectly by decreasing calcium loading and reactive oxygen species (ROS; key inducers of pore opening) or lowering intracellular pH. Agents working in this way may include pyruvate, propofol, Na+/H+ antiporter inhibitors, and ischaemic preconditioning (IPC). Mitochondrial KATP channels have been implicated in preconditioning, but our own data suggest that the channel openers and blockers used in these studies work through alternative mechanisms. In addition to its role in necrosis, transient opening of the MPTP may occur and lead to the release of cytochrome c and other proapoptotic molecules that initiate the apoptotic cascade. However, only if subsequent MPTP closure occurs will ATP levels be maintained, ensuring that cell death continues down an apoptotic, rather than a necrotic, pathway.

First, we present a summary of the evidence for our model of the molecular mechanism of the permeability transition (MPT). Our proposal is that the MPT occurs as a result of the binding of mitochondrial cyclophilin (CyP-D) to the adenine nucleotide translocase (ANT) in the inner mitochondrial membrane. This binding is enhanced by thiol modification of the ANT caused by oxidative stress or other thiol reagents. CyP-D binding enhances the ability of the ANT to undergo a conformational change triggered by Ca2+. Binding of ADP or ATP to a matrix site of the ANT antagonises this effect of Ca2+; modification of other ANT thiol groups inhibits ADP binding and sensitises the MPT to [Ca2+]. Increased membrane potential changes the ANT conformation to enhance ATP binding and hence inhibit the MPT. Our most recent data shows that a fusion protein of CyP-D and glutathione-S-transferase immobilised to Sepharose specifically binds the ANT from Triton-solubilised inner mitochondrial membranes in a cyclosporin A (CsA) sensitive manner. Second we summarise the evidence for the MPT being a major factor in the transition from reversible to irreversible injury during reperfusion of a heart following a period of ischaemia. We describe how in the perfused heart [3H]deoxyglucose entrapment within mitochondria can be used to measure the opening of MPT pore in situ. During ischaemia pore opening does not occur, but significant opening does occur during reperfusion, and recovery of the heart is dependent on subsequent pore closure. Pore opening is inhibited by the presence in the perfusion medium of pyruvate and the anaesthetic propofol which both protect the heart from reperfusion injury. Third we discuss how the MPT may be involved in determining whether cell death occurs by necrosis (extensive pore opening and ATP depletion) or apoptosis (transient pore opening with maintenance of ATP).

Opening of the mitochondrial permeability transition pore (MPTP) is thought to be a critical event in mediating the damage to hearts that accompanies their reperfusion following prolonged ischaemia. Protection from reperfusion injury occurs if the prolonged ischaemic period is preceded by short ischaemic periods followed by recovery. Here we investigate whether such ischaemic preconditioning (IPC) is accompanied by inhibition of MPTP opening. MPTP opening in Langendorff-perfused rat hearts was determined by perfusion with 2-deoxy[ We demonstrate that IPC inhibits initial MPTP opening in hearts reperfused after 30 min global ischaemia, and subsequently enhances pore closure as hearts recover. However, MPTP opening in mitochondria isolated from IPC hearts occurred more readily than control mitochondria, implying that MPTP inhibition by IPC in situ was secondary to other factors such as decreased calcium overload and oxidative stress. Hearts perfused with cyclosporin A or sanglifehrin A, powerful inhibitors of the MPTP, also recovered better from ischaemia than controls (improved haemodynamic function and less lactate dehydrogenase release). However, the mitochondrial DOG entrapment technique showed these agents to be less effective than IPC at preventing MPTP opening. Our data suggest that protection from reperfusion injury is better achieved by reducing factors that induce MPTP opening than by inhibiting the MPTP directly.

Studies with different ATP-sensitive potassium (K ATP ) channel openers and blockers have implicated opening of mitochondrial K ATP (mitoK ATP ) channels in ischaemic preconditioning (IPC). It would be predicted that this should increase mitochondrial matrix volume and hence respiratory chain activity. Here we confirm this directly using mitochondria rapidly isolated from Langendorffperfused hearts. Pre-ischaemic matrix volumes for control and IPC hearts (expressed in ml per mg protein ± S.E.M., n = 6), determined with 3 H 2 O and [14 C]sucrose, were 0.67 ± 0.02 and 0.83 ± 0.04 (P < 0.01), respectively, increasing to 1.01 ± 0.05 and 1.18 ± 0.02 following 30 min ischaemia (P < 0.01) and to 1.21 ± 0.13 and 1.26 ± 0.25 after 30 min reperfusion. Rates of ADP-stimulated (State 3) and uncoupled 2-oxoglutarate and succinate oxidation increased in parallel with matrix volume until maximum rates were reached at volumes of 1.1 ml ml _1 or greater. The mitoK ATP channel opener, diazoxide (50 mM), caused a similar increase in matrix volume, but with inhibition rather than activation of succinate and 2-oxoglutarate oxidation. Direct addition of diazoxide (50 mM) to isolated mitochondria also inhibited State 3 succinate and 2-oxoglutarate oxidation by 30 %, but not that of palmitoyl carnitine. Unexpectedly, treatment of hearts with the mitoK ATP channel blocker 5-hydroxydecanoate (5HD) at 100 or 300 mM, also increased mitochondrial volume and inhibited respiration. In isolated mitochondria, 5HD was rapidly converted to 5HD-CoA by mitochondrial fatty acyl CoA synthetase and acted as a weak substrate or inhibitor of respiration depending on the conditions employed. These data highlight the dangers of using 5HD and diazoxide as specific modulators of mitoK ATP channels in the heart.

In addition to their central role in ATP synthesis, mitochondria play a critical role in cell death. Oxidative stress accompanied by calcium overload, ATP depletion, and elevated phosphate levels induces mitochondrial permeability transition (MPT) with formation of nonspecific MPT pores (MPTP) in the inner mitochondrial membrane. Pore opening results in mitochondrial dysfunction with uncoupled oxidative phosphorylation and ATP hydrolysis, ultimately leading to cell death. For the past 20 years, three proteins have been accepted as key structural components of the MPTP: adenine nucleotide translocase (ANT) in the inner membrane, cyclophilin D (CyP-D) in the matrix, and the voltage-dependent anion channel (VDAC) in the outer membrane. However, most recent studies have questioned the molecular identity of the pores. Genetic studies have eliminated the VDAC as an essential component of MPTP and attributed a regulatory (rather than structural) role to ANT. Currently, the phosphate carrier appears to play a crucial role in MPTP formation. MPTP opening has been examined extensively in cardiac pathological conditions, including ischemia/ reperfusion as well as heart failure. Accordingly, MPTP is accepted as a therapeutic target for both pharmacological and conditional strategies to block pore formation by direct interaction with MPTP components or indirectly by decreasing MPTP inducers. Inhibition of MPTP opening by reduction of CyP-D activity by nonimmunosuppressive analogs of cyclosporine A or sanglifehrin A, as well as attenuation of reactive oxygen species accumulation through mitochondria-targeted antioxidants, is the most promising. This review outlines our current knowledge of the structure and function of the MPTP and describes possible approaches for cardioprotection.Mitochondria play an important role as ATP producers and as regulators of cell death, which make them essential for cell survival. In the heart, mitochondria occupy approximately 30% of cardiomyocyte volume and provide more than 90% ATP necessary for cardiac function. One of the key factors regulating mitochondrial function and ATP synthesis is the mitochondrial Ca 2ϩ concentration. Cardiomyocyte Ca 2ϩ homeostasis is altered under pathological conditions, such as ischemia and heart failure (HF), due to decreased ATP levels resulting from inadequate oxygen consumption. Furthermore, dysfunction of the electron transport chain, particularly during reperfusion, results in increased generation of ROS. Calcium overload and oxidative stress combine with other factors, including high phosphate and low adenine nucleotide concentrations to induce the formation of nonspecific mitochondrial permeability transition pores (MPTP) in the inner mitochondrial membrane (Bernardi et al., 1992;Crompton, 1999;Halestrap et al., 2004). Opening of MPTP causes uncoupling of the mitochondria and swelling of the matrix leading to rupture of the outer mitochondrial membrane and ultimately cell death. In recent years, MPTP openThis work was supported by the Canadian Ins...