Samantha J. Clarke

Mitochondrial permeability transition pore opening during myocardial reperfusion: a target for cardioprotection

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.

The permeability transition pore complex: another view

Mitochondria play a critical role in initiating both apoptotic and necrotic cell death. A major player in this process is the mitochondrial permeability transition pore (MPTP), a non-specific pore, permeant to any molecule of < 1.5 kDa, that opens in the inner mitochondrial membrane under conditions of elevated matrix [Ca(2+)], especially when this is accompanied by oxidative stress and depleted adenine nucleotides. Opening of the MPTP causes massive swelling of mitochondria, rupture of the outer membrane and release of intermembrane components that induce apoptosis. In addition mitochondria become depolarised causing inhibition of oxidative phosphorylation and stimulation of ATP hydrolysis. Pore opening is inhibited by cyclosporin A analogues with the same affinity as they inhibit the peptidyl-prolyl cis-trans isomerase activity of mitochondrial cyclophilin (CyP-D). These data and the observation that different ligands of the adenine nucleotide translocase (ANT) can either stimulate or inhibit pore opening led to the proposal that the MPTP is formed by a Ca-triggered conformational change of the ANT that is facilitated by the binding of CyP-D. Our model is able to explain the mode of action of a wide range of known modulators of the MPTP that exert their effects by changing the binding affinity of the ANT for CyP-D, Ca(2+) or adenine nucleotides. The extensive evidence for this model from our own and other laboratories is presented, including reconstitution studies that demonstrate the minimum configuration of the MPTP to require neither the voltage activated anion channel (VDAC or porin) nor any other outer membrane protein. However, other proteins including Bcl-2, BAX and virus-derived proteins may interact with the ANT to regulate the MPTP. Recent data suggest that oxidative cross-linking of two matrix facing cysteine residues on the ANT (Cys(56) and Cys(159)) plays a key role in regulating the MPTP. Adenine nucleotide binding to the ANT is inhibited by Cys(159) modification whilst oxidation of Cys(56) increases CyP-D binding to the ANT, probably at Pro(61).

Ischaemic preconditioning inhibits opening of mitochondrial permeability transition pores in the reperfused rat heart

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[3H]glucose ([3H]DOG) and measurement of mitochondrial [3H]DOG entrapment. 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.

Sanglifehrin A Acts as a Potent Inhibitor of the Mitochondrial Permeability Transition and Reperfusion Injury of the Heart by Binding to Cyclophilin-D at a Different Site from Cyclosporin A

Cyclosporin A (CsA) inhibits opening of the mitochondrial permeability transition pore (MPTP), a critical event in some forms of necrotic and apoptotic cell death, by binding to cyclophilin D (CyP-D) and inhibiting its peptidyl-prolylcis-trans isomerase (PPIase) activity. Sanglifehrin A (SfA), like CsA, exerts its immunosuppressive action by binding to cyclophilin A but at a different site from CsA, and unlike the latter, SfA does not inhibit calcineurin activity. Here we demonstrate that SfA inhibits the PPIase activity of CyP-D (K 0.5 2 nm) and acts as a potent inhibitor of MPTP opening under both energized and de-energized conditions. However, unlike CsA, the dose-response curve for inhibition by SfA is sigmoidal rather than hyperbolic, suggesting a multimeric structure for the MPTP with cooperativity between subunits. Furthermore, SfA does not prevent CyP-D binding to submitochondrial particles or detergent-solubilized adenine nucleotide translocase (ANT), implying that CyP-D binding to the ANT does not require PPIase activity but pore opening does. Once bound to the MPTP, SfA is not readily dissociated, and inhibition of pore opening is maintained following extensive washing. To investigate the potential of SfA as an inhibitor of cell death in vivo, we used the Langendorff perfused rat heart. SfA caused a time-dependent inhibition of the MPTP that was maintained on mitochondrial isolation to a greater extent than was CsA inhibition. We demonstrate that SfA, like CsA, improves the recovery of left ventricular developed pressure during reperfusion after 30 min of global ischemia and greatly reduces lactate dehydrogenase release, implying inhibition of necrotic damage. Because SfA does not inhibit calcineurin activity, our data suggest that it may be more desirable than CsA for protecting tissues recovering from ischemic episodes and for studying the role of the MPTP in cell death.

The effects of ischaemic preconditioning, diazoxide and 5-hydroxydecanoate on rat heart mitochondrial volume and respiration

Studies with different ATP‐sensitive potassium (KATP) channel openers and blockers have implicated opening of mitochondrial KATP (mitoKATP) 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 Langendorff‐perfused hearts. Pre‐ischaemic matrix volumes for control and IPC hearts (expressed in μl per mg protein ±s.e.m., n= 6), determined with 3H2O and [14C]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 μl ml−1 or greater. The mitoKATP channel opener, diazoxide (50 μm), caused a similar increase in matrix volume, but with inhibition rather than activation of succinate and 2‐oxoglutarate oxidation. Direct addition of diazoxide (50 μm) 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 mitoKATP channel blocker 5‐hydroxydecanoate (5HD) at 100 or 300 μm, 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 mitoKATP channels in the heart.

Inhibition of Mitochondrial Permeability Transition Pore Opening by Ischemic Preconditioning Is Probably Mediated by Reduction of Oxidative Stress Rather Than Mitochondrial Protein Phosphorylation

Inhibition of mitochondrial permeability transition pore (MPTP) opening at reperfusion is critical for cardioprotection by ischemic preconditioning (IP). Some studies have implicated mitochondrial protein phosphorylation in this effect. Here we confirm that mitochondria rapidly isolated from preischemic control and IP hearts show no significant difference in calcium-mediated MPTP opening, whereas IP inhibits MPTP opening in mitochondria isolated from IP hearts following 30 minutes of global normothermic ischemia or 3 minutes of reperfusion. Analysis of protein phosphorylation in density-gradient purified mitochondria was performed using both 2D and 1D electrophoresis, with detection of phosphoproteins using Pro-Q Diamond or phospho-amino-specific antibodies. Several phosphoproteins were detected, including voltage-dependent anion channels isoforms 1 and 2, but none showed significant IP-mediated changes either before ischemia or during ischemia and reperfusion, and neither Western blotting nor 2D fluorescence difference gel electrophoresis detected translocation of protein kinase C (&agr;, ϵ, or &dgr; isoforms), glycogen synthase kinase 3&bgr;, or Akt to the mitochondria following IP. In freeze-clamped hearts, changes in phosphorylation of GSK3&bgr;, Akt, and AMP-activated protein kinase were detected following ischemia and reperfusion but no IP-mediated changes correlated with MPTP inhibition or cardioprotection. However, measurement of mitochondrial protein carbonylation, a surrogate marker for oxidative stress, suggested that a reduction in mitochondrial oxidative stress at the end of ischemia and during reperfusion may account for IP-mediated inhibition of MPTP. The signaling pathways mediating this effect and maintaining it during reperfusion are discussed.

Temperature preconditioning of isolated rat hearts – a potent cardioprotective mechanism involving a reduction in oxidative stress and inhibition of the mitochondrial permeability transition pore

We investigate whether temperature preconditioning (TP), induced by short‐term hypothermic perfusion and rewarming, may protect hearts against ischaemic/reperfusion injury like ischaemic preconditioning (IP). Isolated rat hearts were perfused for 40 min, followed by 25 min global ischaemia and 60 min reperfusion (37°C). During pre‐ischaemia, IP hearts underwent three cycles of 2 min global ischaemia and 3 min reperfusion at 37°C, whereas TP hearts received three cycles of 2 min hypothermic perfusion (26°C) interspersed by 3 min normothermic perfusion. Other hearts received a single 6 min hypothermic perfusion (SHP) before ischaemia. Both IP and TP protocols increased levels of high energy phosphates in the pre‐ischaemic heart. During reperfusion, TP improved haemodynamic recovery, decreased arrhythmias and reduced necrotic damage (lactate dehydrogenase release) more than IP or SHP. Measurements of tissue NAD+ levels and calcium‐induced swelling of mitochondria isolated at 3 min reperfusion were consistent with greater inhibition of the mitochondrial permeability transition at reperfusion by TP than IP; this correlated with decreased protein carbonylation, a surrogate marker for oxidative stress. TP increased protein kinase Cɛ (PKCɛ) translocation to the particulate fraction and pretreatment with chelerythrine (PKC inhibitor) blocked the protective effect of TP. TP also increased phosphorylation of AMP‐activated protein kinase (AMPK) after 5 min index ischaemia, but not before ischaemia. Compound C (AMPK inhibitor) partially blocked cardioprotection by TP, suggesting that both PKC and AMPK may mediate the effects of TP. The presence of N‐(2‐mercaptopropionyl) glycine during TP also abolished cardioprotection, indicating an involvement of free radicals in the signalling mechanism.