Does the Mitochondrial Permeability Transition Have a Role in Preconditioning?
To the Editor:
In a recent Brief Rapid Communication, Hausenloy et al1 implicate transient opening of the mitochondrial permeability transition pore (mPTP) in mediating ischemic preconditioning (IPC) and preconditioning induced by uncoupler and diazoxide. Their most persuasive evidence is the ability of cyclosporin A (CsA) and sanglifehrin A (SfA), both potent mPTP inhibitors, to overcome the protection when added during the preconditioning phase. However, there are reasons to question the authors’ conclusions.
First, although not mentioned by the authors, our own data using the mitochondrial 2-deoxyglucose entrapment technique failed to detect any IPC-induced increase in mPTP opening in the perfused heart, although, as predicted, less mPTP opening was observed in IPC hearts after 30 minutes of ischemia and reperfusion.2 This inhibitory effect is probably mediated indirectly through decreased calcium overload or oxidative stress at reperfusion.2
Second, the authors suggest that transient mPTP opening during the preconditioning phase prevents mitochondrial calcium overload and that this mediates protection. However, calcium overload occurs at reperfusion and not during the preischemic phase when preconditioning occurs. Such mitochondrial calcium overload at reperfusion, together with oxidative stress, causes opening of the mPTP that compromises ATP production, leading to loss of ionic homeostasis and hence necrotic cell death. Indeed, as demonstrated in this laboratory and by the authors themselves, inhibiting mPTP opening at reperfusion with CsA or SfA protects hearts from reperfusion injury.3 This makes the ability of SfA and CsA to antagonize preconditioning somewhat surprising, especially given that these agents bind extremely tightly to mitochondria and remain attached even with extensive washing.2 Thus, even though removed from the perfusion, they might be expected to continue to inhibit the mPTP during ischemia and reperfusion.
Third, when using an uncoupler to precondition, the authors argue that this enhances mPTP opening. However, uncoupling depolarizes mitochondria independently of any mPTP, opening and this will decrease their calcium loading just as will mPTP opening. However, in this case, the response will be insensitive to either SfA or CsA. Yet the authors show that both CsA and SfA overcome the protection mediated by uncoupler implying that these agents are acting independently of mPTP opening.
In conclusion, attractive though the authors’ hypothesis is, we should not rule out an alternative explanation of their data. This is likely to involve roles for cyclophilins in the preconditioning signaling pathway, perhaps similar to those involved in the immunosuppressive actions of these drugs.
Hausenloy D, Wynne A, Duchen M, Yellon D. Transient mitochondrial permeability transition pore opening mediates preconditioning-induced protection. Circulation. 2004; 109: 1714–1717.
Halestrap AP, Clarke SJ, Javadov SA. Mitochondrial permeability transition pore opening during myocardial reperfusion: a target for cardioprotection. Cardiovasc Res. 2004; 61: 372–385.
The data published in our recent Brief Rapid Communication1 implicated transient opening of the mitochondrial permeability transition pore (mPTP) as a mediator of the cardioprotection induced by ischemic preconditioning (IPC), diazoxide, CCPA (the adenosine A1-receptor agonist) and a mitochondrial uncoupler. In all cases, preconditioning was abrogated by exposure of the heart to mPTP inhibitors given at the time of preconditioning.
We would like to respond to Dr Halestrap’s recent letter, in which he questioned the interpretation of these observations.
1. Dr Halestrap states that, using the mitochondrial 2-deoxyglucose entrapment technique, his group did not observe an IPC-induced increase in mPTP opening.2 However, in their study,2 they did note an IPC-induced increase in mPTP opening in mitochondria isolated immediately after the IPC protocol, when using an alternative technique for detecting mPTP opening.
2. We speculated1 that transient mPTP opening may mediate protection by attenuating mitochondrial calcium loading prior to ischemia, thereby rendering mitochondria more resistant to the mitochondrial calcium loading at reperfusion, and thus suppress mPTP opening. Obviously, mitochondrial calcium loading during ischemia-reperfusion is the critical mediator of mPTP opening at reperfusion.
3. Dr Halestrap expresses surprise that the mPTP inhibitors, which were cardioprotective when given at reperfusion,3 also abrogate IPC-induced protection when given before ischemia. Our data suggest a dichotomous role for the mPTP, in which transient mPTP opening before ischemia mediates IPC-induced protection,1 whereas IPC in turn protects by inhibiting mPTP opening at reperfusion.2,3 This scenario echoes the role of reactive oxygen species (ROS) in IPC; ROS produced before ischemia mediate IPC-induced protection,4 whereas ROS produced at reperfusion increase cellular injury, and IPC attenuates ROS production at reperfusion.5
4. Dr Halestrap argues that the mPTP inhibitors abrogated uncoupling-induced protection independent of any effect on the mPTP. We propose that uncoupler-mediated depolarization triggers mPTP opening, as the latter is voltage-sensitive, explaining why uncoupling-induced protection was prevented by mPTP inhibitors.1
The data published in our communication1 argue strongly that transient mPTP opening may act to mediate cardioprotection in response to a variety of preconditioning stimuli.
Hausenloy D, Wynne A, Duchen M, et al. Transient mitochondrial permeability transition pore opening mediates preconditioning-induced protection. Circulation. 2004; 109: 1714–1717.
Hausenloy DJ, Maddock HL, Baxter GF, et al. Inhibiting mitochondrial permeability transition pore opening: a new paradigm for myocardial preconditioning? Cardiovasc Res. 2002; 55: 534–543.
Vanden Hoek TL, Becker LB, Shao Z, et al. Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes. J Biol Chem. 1998; 273: 18092–18098.
Vanden Hoek T, Becker LB, Shao ZH, et al. Preconditioning in cardiomyocytes protects by attenuating oxidant stress at reperfusion. Circ Res. 2000; 86: 541–548.