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

Although resveratrol has been demonstrated to be cardioprotective, the detailed cellular and molecular mechanisms that mediate the protection remain elusive. We aimed to determine if resveratrol protects the heart at reperfusion by modulating the mitochondrial permeability transition pore (mPTP) opening through glycogen synthase kinase 3β (GSK-3β). Resveratrol (10μM) given at reperfusion reduced infarct size (12.2 ± 2.5 % of risk zone vs. 37.9 ± 3.1 % of risk zone in control, n = 6) in isolated rat hearts subjected to 30 min regional ischemia followed by 2 h of reperfusion, an effect that was abrogated by the mPTP opener atractyloside (30.9 ± 8.1 % of risk zone), implying that resveratrol may protect the heart at reperfusion by modulating the mPTP opening. To define the signaling mechanism underlying the action of resveratrol, we determined GSK-3β activity by measuring its phosphorylation at Ser 9 . Resveratrol significantly enhanced GSK-3β phosphorylation upon reperfusion (225.2 ± 30.0 % of control at 5 min of reperfusion). Further experiments showed that resveratrol induces translocation of GSK-3β to mitochondria and translocated GSK-3β interacts with the mPTP component cyclophilin D but not VDAC (the voltage-dependent anion channel) or ANT (the adenine nucleotide translocator) in cardiac mitochondria. Taken together, these data suggest that resveratrol prevents myocardial reperfusion injury by targeting the mPTP opening via GSK-3β. Translocation of GSK-3β to mitochondria and its interaction with the mPTP component cyclophilin D may serve as an essential mechanism that mediates the protective effect of resveratrol on reperfusion injury.

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The mitochondrial permeability transition: its molecular mechanism and role in reperfusion injury

Biochemical Society Symposium ◽

10.1042/bss0660181 ◽

1999 ◽

Vol 66 ◽

pp. 181-203 ◽

Cited By ~ 125

Author(s):

Andrew P. Halestrap

Keyword(s):

Oxidative Stress ◽

Cyclosporin A ◽

Reperfusion Injury ◽

Molecular Mechanism ◽

Mitochondrial Permeability Transition ◽

Adenine Nucleotide ◽

Apoptotic Cell ◽

Heart Function ◽

Permeability Transition ◽

Mitochondrial Permeability

The mitochondrial permeability transition (mPT) involves the opening of a non-specific pore in the inner membrane of mitochondria, converting them from organelles whose production of ATP sustains the cell, to instruments of death. Here, I first summarize the evidence in favour of our model for the molecular mechanism of the mPT. It is proposed that the adenine nucleotide translocase (ANT) is converted into a non-specific pore through a calcium-mediated conformational change. This requires the binding of a unique cyclophilin (cyclophilin-D, CyP-D) to the ANT, except when matrix [Ca2+] is very high. Binding of CyP-D is increased in response to oxidative stress and some thiol reagents which sensitize the mPT to [Ca2+]. Matrix adenine nucleotides decrease the sensitivity of the mPT to [Ca2+] by binding to the ANT. This is antagonized by carboxyatractyloside (an inhibitor of the ANT) and by modification of specific thiol groups on the ANT by oxidative stress or thiol reagents; such treatments thus enhance the mPT. In contrast, decreasing intracellular pH below 7.0 greatly desensitizes the mPT to [Ca2+]. Conditions which sensitize the mPT towards [Ca2+] are found in hearts reperfused after a period of ischaemia, a process that may irreversibly damage the heart (reperfusion injury). We have demonstrated directly that mPT pores open during reperfusion (but not ischaemia) using a technique that involves entrapment of [3H]deoxyglucose in mitochondria that have undergone the mPT. The mPT may subsequently reverse in hearts that recover from ischaemia/reperfusion, the extent of resealing correlating with recovery of heart function. A variety of agents that antagonize the mPT protect the heart from reperfusion injury, including cyclosporin A, pyruvate and propofol. Mitochondria that undergo the mPT and then reseal may cause cytochrome c release and thus initiate apoptosis in cells subjected to stresses less severe than those causing necrosis. An example is the apoptotic cell death in the hippocampus that occurs several days after insulin-induced hypoglycaemia, and can be prevented by prior treatment with cyclosporin A.

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Phosphate is not an absolute requirement for the inhibitory effects of cyclosporin A or cyclophilin D deletion on mitochondrial permeability transition

Biochemical Journal ◽

10.1042/bj20111881 ◽

2012 ◽

Vol 443 (1) ◽

pp. 185-191 ◽

Cited By ~ 10

Author(s):

Allison M. McGee ◽

Christopher P. Baines

Keyword(s):

Cyclosporin A ◽

Mitochondrial Permeability Transition ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Liver Mitochondria ◽

Cyclophilin D ◽

Wild Type ◽

Inhibitory Effects ◽

Mitochondrial Permeability ◽

Pre Treatment

CypD (cyclophilin D) has been established as a critical regulator of the MPT (mitochondrial permeability transition) pore, and pharmacological or genetic inhibition of CypD attenuates MPT in numerous systems. However, it has recently been suggested that the inhibitory effects of CypD inhibition only manifest when Pi (inorganic phosphate) is present, and that inhibition is lost when Pi is replaced by Asi (inorganic arsenate) or Vi (inorganic vanadate). To test this, liver mitochondria were isolated from wild-type and CypD-deficient (Ppif−/−) mice and then incubated in buffer containing Pi, Asi or Vi. MPT was induced under both energized and de-energized conditions by the addition of Ca2+, and the resultant mitochondrial swelling was measured spectrophotometrically. For pharmacological inhibition of CypD, wild-type mitochondria were pre-incubated with CsA (cyclosporin A) before the addition of Ca2+. In energized and de-energized mitochondria, Ca2+ induced MPT regardless of the anion present, although the magnitude differed between Pi, Asi and Vi. However, in all cases, pre-treatment with CsA significantly inhibited MPT. Moreover, these effects were independent of mouse strain, organ type and rodent species. Similarly, attenuation of Ca2+-induced MPT in the Ppif−/− mitochondria was still observed irrespective of whether Pi, Asi or Vi was present. We conclude that the pharmacological and genetic inhibition of CypD is still able to attenuate MPT even in the absence of Pi.

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