Presentation on theme: "Andrew Halestrap Calcium, mitochondria and reperfusion injury"— Presentation transcript:
1 Andrew Halestrap Calcium, mitochondria and reperfusion injury A pore way to dieAndrew HalestrapDepartment of BiochemistryandThe Bristol Heart Institute
2 X ATP synthesis Cell lives Cell dies ATP breakdown The Mitochondrial Permeability TransitionA calcium induced non-specific poreCyclosporin AXAll solutes < 1500daltonsMitochondrial innermembraneVery selective permeability essential for ATP synthesisMitochondria become leaky, swollen and uncoupledATP synthesisCell livesCell diesATP breakdownIf the pore opens, not only are mitochondria unable to make ATP, they also breakdown ATP made by functional mitochondria and glycolysis.Triggered by high matrix [Ca ]2+Sensitised to Ca by:Low adenine nucleotidesHigh phosphateOxidative stressConditions occurring when cells damaged e.g. by toxins or hypoxia
3 Proposed scheme for the mechanism of pore opening Normal Impermeable StateCaTriggered by low [Ca 2+]Pathological Non-specific PoreCa2+CytosolADPAdeninenucleotidetranslocaseATPMatrixBinding increased by oxidative stress and thiol reagents. CyP binding increases sensitivity to [Ca].ATPADPImpermeableStateCyclophilin DActivated by thiol reagents and oxidative stress which decrease ADP/ATP bindingInhibited by [Mg 2+], low pH, adenine nucleotides and membrane potential(which increases ATP binding)Cyclosporin ANote that outer membrane proteins such as VDAC (porin), Bcl2 family members and the peripheral benzodiazipine receptor may be involved as regulatory or structural components
4 Evidence for the involvement of Cyclophilin D For all CsA analogues tested the K0.5 for inhibition of the peptidyl-prolyl cis-trans isomerase activity of CyP-D correlates with the K0.5 for inhibition of the MPTPThe number of binding sites for CsA inhibition of the MPTP matches the number of binding sites for CsA inhibition of the PPIase activity of the mitochondrial matrix
5 CyP-D knockout mice show impaired permeability transition Baines, C. P., Kaiser, R. A., Purcell, N. H., Blair, N. S., Osinska, H., Hambleton, M. A., Brunskill, E. W., Sayen, M. R., Gottlieb, R. A., Dorn, G. W., Robbins, J., and Molkentin, J. D. (2005). Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature, 434:Basso, E., Fante, L., Fowlkes, J., Petronilli, V., Forte, M. A., and Bernardi, P. (2005). Properties of the permeability transition pore in mitochondria devoid of cyclophilin D. J Biol Chem, 280:Nakagawa, T., Shimizu, S., Watanabe, T., Yamaguchi, O., Otsu, K., Yamagata, H., Inohara, H., Kubo, T., and Tsujimoto, Y. (2005). Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature, 434:Schinzel, A. C., Takeuchi, O., Huang, Z., Fisher, J. K., Zhou, Z., Rubens, J., Hetz, C., Danial, N. N., Moskowitz, M. A., and Korsmeyer, S. J. (2005). Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia. Proc Natl Acad Sci U S A, 102:
6 The MPTP opens in CyP-D knockout mitochondria at high calcium loading
7 .s .10 ) Rate of shrinkage (A [Ca Inhibition of the permeability transition by CsA and SfA is overcome by high [Ca2+]2468[Ca+]mM1357RteofShrinkg(A.s-x)ControlSfA10203040506070Rate of shrinkage (A520.s )80[Ca2+Shrinkage1 mM SfAor 1 mM CsA
8 Proposed scheme for the mechanism of pore opening Normal Impermeable StateCaTriggered by low [Ca 2+]Pathological Non-specific PoreCa2+CytosolCaVery high [Ca2+]ADPAdeninenucleotidetranslocaseATPMatrixBinding increased by oxidative stress and thiol reagents. CyP binding increases sensitivity to [Ca].ATPADPImpermeableStateCyclophilin DActivated by thiol reagents and oxidative stress which decrease ADP/ATP bindingInhibited by [Mg 2+], low pH, adenine nucleotides and membrane potential(which increases ATP binding)Cyclosporin A
9 The Immunosuppressive drug Sanglifehrin A is a novel inhibitor of the MPTP This inhibitor is Sanglifehrin A.Sanglifehrin A is and has a very different structure from Cyclosporin A.In terms of its properties:Sanglifehrin A is similar to cyclosporin A in that it binds tightly to cyclophilin A.Although it has immunosuppressive properties (by blocking T-cell proliferation in response to interleukin-2),unlike CsA, the cyclophilin-Sanglifehrin complex has no effect on the calcium-dependent protein phosphatase, calcineurin which mediates the immunosuppressant activity of CsA.CLICKCyclosporin ASanglifehrin ALike CsA, SfA also binds tightly to CyP- A (K nM)Unlike CsA, the CyPA-SfA complex has no effect on calcineurin
10 SfA inhibits the peptidyl prolyl cis trans isomerase activity of CyP-D and inhibits the permeability transition)616-s(30 sA5200.05Control150 nM SfA150 nM CsA500 nM SfA1 mM SfA1 mM CsA500 nM CsACa2+De-energised swellings-1is5PPiase activity of CyP-Dy5lordy4h4peptide hydrolysis (s )Rate constant foredti3p3eKi 2nMprof22tnats1n1ocetaR51015202510051152251[Sanglifehrin] (nM)[Sanglfehrin]nM
12 Proposed scheme for the mechanism of pore opening Normal Impermeable StateCaTriggered by low [Ca 2+]Pathological Non-specific PoreCa2+CytosolCaVery high [Ca2+]ADPAdeninenucleotidetranslocaseATPMatrixBinding increased by oxidative stress and thiol reagents. CyP binding increases sensitivity to [Ca].ATPADPImpermeableStateCyclophilin DActivated by thiol reagents and oxidative stress which decrease ADP/ATP bindingInhibited by [Mg 2+], low pH, adenine nucleotides and membrane potential(which increases ATP binding)Sanglifehrin ASfAXCyclosporin A
13 Evidence for the involvement of the Adenine Nucleotide Translocase
14 Inhibition of the MPT by ADP 51[Ca2+]mMRteofshrinkg(A.-4)DPTlBK751234[Ca+]mMRteofShrinkg(A.s-x)lDP
15 The ANT binds to immobilised CyP-D in a CsA sensitive manner Western blotsNo IMMCsA + IMMCsH + IMMPlus IMMPorinGlutathione eluted proteinsCyclophilin affinity columnMatrixGSHGSTCyP-DAdd Triton-X100-solubilised innermitochondrial membranesWash off non-specifically bound proteinGSHSpecifically boundprotein30kDaControlDiamideMore ANT from diamide-treated mitochondria binds GST-CyP-D columnSDS-PAGE and Western blottingwith anti ANT antibodies
16 Inhibition of the MPT by ADP is antagonised by thiol modification
17 Pro Cys Cys Cys Location in the ANT of residues with potential regulatory significance for the MPTProADPCys61LowN-ethylmaleimide56bindingCysEosine-Cys159256maleimide and high NEMCyPbinding?
18 Eosine maleimide (to block Cys159) blocks ATP inhibition of the MPTP 24681[ADP]mMRateofshrinkg(5.-)EldNC+Cys159Cys56Cys56 plus some Cys159
19 Pro Cys Cys Cys Location in the ANT of residues with potential regulatory significance for the MPTProADP61N-ethyl-maleimideEosine-Cys56bindingCysCys159256Diamide and Phenylarsine oxide crosslinks Cys159to Cys256CyPbinding?
20 Section through the carrier Section through the carrier. At the bottom of the cone-shaped cavity, the hexapeptide (RRMMM signature) can be seen. The conical pit open to the outside and the RRR sequence spanning through the closed part of the carrier.Nature 426, (06 November 2003)Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractylosideEVA PEBAY-PEYROULA1, CÉCILE DAHOUT-GONZALEZ2, RICHARD KAHN1, VÉRONIQUE TRÉZÉGUET3, GUY J.-M. LAUQUIN3 & GÉRARD BRANDOLIN2
21 Nature 427, (29 January 2004);The ADP/ATP translocator is not essential for the mitochondrial permeability transition poreJASON E. KOKOSZKA1,4,*, KATRINA G. WAYMIRE1,4, SHAWN E. LEVY4,*, JAMES E. SLIGH4,*, JIYANG CAI5, DEAN P. JONES5, GRANT R. MACGREGOR1,2,4 & DOUGLAS C. WALLACE1,3,4Key point:Mice with a double ANT knockout in their liver mitochondria still show a cyclosporin-sensitive MPTP. However, it is less sensitive to Ca2+ and is not blocked by ADP or activated by CAT
22 Some interesting questions 1. How can mice survive with a liver that lacks ANT? Mitochondrial export of ATP is essential for gluconeogenesis and urea synthesis for example. Do the mice have complete liver ANT knockout?2. Even if the ANT is not functioning as the MPTP pore in the knockout, it may still do so in the normal mouse.Can other members of the mitochondrial carrier family, present in MUCH lower quantity than the ANT, act as the CyP-D binding membrane pore structure if ANT is lacking?
23 NADH NAD+ NADH OAA A OAA MAL X CyP-D ANT MPT Ca MDH MDH Pore opens A 340OAAMALXANTPorinSilver stainHAHiTCyP-DANTMPTCa2+NADHNAD+OAAMALMDHPore opensMDHCyP-DCyP-D + CsA7.52862250mM Ca6.5A3400.023 minCsA presentin bufferCsAnotpresent inbuffer2 minA3400.01120mM Ca2g/ml CyP-D2+Data of Jeremy Gillespie
25 Reversible reperfusion injury Irreversible reperfusion injury Ischaemia (major reduction or total loss of blood flow)ATP drops and cell begins to deteriorate as a result of metabolic and ionicdisturbances (e.g. lactic acid build up, low pH, high [Na] and [Ca], free radicals)Short period of ischaemiaMild to moderate damageReversible on reperfusionReversible reperfusion injury"Stunned" heart eventuallyrecovers fullyLong period of ischaemiaSevere damage exacerbatedon reperfusion which leads tocell death and necrosisAssociated with swollen amorphousmitochondria that are uncoupled andhave impaired respiration.They look as if the pore has opened.Irreversible reperfusion injury
26 Repair of damage Cell survives Further damage Cell dies Ischaemia / ReperfusionATP depletionDisruption of ionic homeostasisCalcium overloadOxidative StressDamage to intracellular componentsMPTP closed - High ATPLow ATP – MPTP openRepair of damage Cell survivesFurther damage Cell diesMitochondriaLIFEDEATHJudge and executioner
27 The "Hot DOG" technique for measuring pore opening [ H]-DOG (2-deoxyglucose)3Load heart cells with 2-deoxyglucose(DOG) which is trapped in the cytosol as DOG-6-PDOGDOG-6-PDOG-6-PXPore closedPore openDOG-6-P only enters mitochondria if pore opensThe amount of [3H]-DOG-6-P in mitochondria is used as an indicator of pore opening. Corrections are made variations in cell loading with DOG (measure whole tissue DOG) and mitochondrial recovery (citrate synthase)Isolate mitochondria in EGTA buffer. Open pores rapidly close and trap DOG+ DOG-6-P in matrix.t (min)ISCHAEMIA2010050601253H-DOG-loadingIsolated mitochondria
28 Pore opens as pH returns to normal. (Pore is inhibited at pH<7.0) Time dependence of mitochondrial pore opening and pH recovery during reperfusion of hearts after 30 min ischaemiapHDOGPore opens as pH returns to normal. (Pore is inhibited at pH<7.0)30 min ischemiaPITime of postischaemic reperfusion (min)- 0- 100mm HgIschaemiaData of Paul Kerr
29 Does prevention of MPTP opening protect hearts from reperfusion injury?
30 Ischaemic Preconditioning Protecting hearts from reperfusion injury(Cyclosporin A, Sanglifehrin A and CyP-D knockout)Direct inhibitors of the MPTPPyruvateLess oxidative stress(Free radical scavengers e.g. propofol)Low pH and [Ca2+]mitoIschaemic Preconditioning
31 Cyclosporin A and Sanglifehrin A protects hearts from reperfusion injury Data of Sam Clarke and Gavin McStayLVDPLVEDPConSfACsA80604010020LVDP (% preischaemic value)LEVDP (mm Hg)ControlSfACsA30408085909510010511030 min Ischaemia60501020Time (min)*LDH releaseLDH released (munits/ml perfusate)
32 Baines et al (2005). Nature, 434: 658-662. The hearts of CyP-D knockout mice are protected from reperfusion injuryBaines et al (2005). Nature, 434:Nakagawa et al (2005).. Nature, 434:
33 Cyclosporin protects neurons against hypoglycaemic and ischaemic damage Friberg, H.; FerrandDrake, M.; Bengtsson, F.; Halestrap, A. P., Wieloch, T. (1998) Cyclosporin A, but not FK 506, protects mitochondria and neurons against hypoglycemic damage and implicates the mitochondrial permeability transition in cell death. J Neurosci 18:Matsumoto, S.; Friberg, H.; FerrandDrake, M., Wieloch, T. (1999) Blockade of the mitochondrial permeability transition pore diminishes infarct size in the rat after transient middle cerebral artery occlusion. J Cerebral Blood Flow Metab 19:Toluidine bluestainedControl30 min insulinHypoglycaemia30 min + CsAHypoglycaemiaControl30 min insulinHypoglycaemia30 min + CsA
34 Sensitivity of MPT to [Ca] in mitochondria from different regions of the brain CerebellumCortexHippocampus[Ca2+] mM1005020Rate of swelling150250200100 mM Ca2mM EGTACerebellumA520Hippocampus1minFriberg, H.; Connern, C.; Halestrap, A. P., and Wieloch, T. (1999) J. Neurochem. 72:
36 Does this involve inhibition of pore opening? Ischaemic PreconditioningBrief ischaemic episodes followed by recovery protect the heart against subsequent prolonged ischaemia and reperfusion.Does this involve inhibition of pore opening?
37 Data of Sabzali Javadov and Kelvin Lim Protection of hearts from reperfusion injury by ischemic preconditioning** p<0.01Data of Sabzali Javadov and Kelvin Lim
39 Pre-ischaemia End of Ischemia 3 min Reperfusion Ischaemic preconditioning decreases Ca2+-dependent swelling of mitochondria isolated following Ischaemia/Reperfusion7Control6IP5** P<0.01vs. ControlA4**32**1Pre-ischaemia End of Ischemia 3 min ReperfusionRapidly isolated mitochondria incubated under de-energised conditions in the presence of a calcium ionophore with swelling initiated by addition of 50 mM Ca2+Data of Igor Khaliulin, Sam Clarke and Jo Parker
40 Pyruvate protects hearts from reperfusion injury Control ischemiaPyruvate-treated ischemia36.2± 9.9Control40LVDP% control(6)10 mM Pyruvate105.3± 17.2(5)(4)Post-ischemicDOG loading57.2± 10.398.9± 10.8ISCHAEMIA10050601253H-DOG-loading+/- 10 mM pyruvate155165t (min)Post-ischaemic loadingPyruvate is:a free radical scavengera good respiratory substrate for ATP productionIt causes acidification30(4)Mitochondrial DOG uptake (Ratio units)20Pre-ischaemic loading(4)+/- 10 mM pyruvate205060100125103H-DOG-loadingISCHAEMIAt (min)NoPre-ischemicischemiaDOG loadingData of Paul Kerr
41 Effects of PROPOFOL on mitochondrial pore opening and functional recovery during reperfusion of hearts after 30 min ischaemiaControlPropofol510152025MPTP opening in situ (DOG units)406080Rate of mitochondrial swelling at 100mM Ca2+DOGSwellingRate of mitochondrial swelling at 100 mM Ca2+LVDPEDP706050LVDP and EDP (mm Hg)40302010Data of Sabzali Javadov
45 The role of mitochondria in initiating the apoptotic cascade t-BidBcl-2ApoptoticsignalActivation ofApaf-1NucleareffectsBax+-MPTPApaf-1Cyt-cpro-Caspase-9dATPAIFSmacIMMOMMMitochondrionActivation ofapoptoticcascadecleavageCaspase-9pro-Caspase-3Caspase-3Proteolytic-CaspaseinhibitorproteinsMPTP opening causes swelling and release of intermembrane proteins. Could MPTP opening be involved in apoptosis?
46 Stress to cell - ? bcl-2 APOPTOSIS NECROSIS Mitochondrial Permeability TransitionMitochondrial swelling and outer membrane ruptureRelease of cytochrome c, AIF and Smac/DiabloActivation of caspasesAPOPTOSISMPT pores closeATP production maintainedModerate insultpores resealThe extent of the permeability transition may determine whether cell death is necrotic or apoptotic following ischaemia and reperfusionNECROSISMPT pores stay openATP is depletedSevere insultpores remain open
47 ? - - Role of the Mitochondrial Permeability Transition in Apoptosis and Necrosisbcl-2-Removal of growth factorsCytokines e.g. TNF, FasActivation of Caspase 8?t-Bid and Bax migrate tomitochondrial outermembraneMultiple and interacting signalling pathwaysStresses e.g. reperfusion or toxinsMitochondrial Permeability TransitionSwelling and outer membrane ruptureTransient opening-?bcl-2Release of cytochrome c,Activation ofActivation ofSmac/Diablo and AIFcaspase 3caspase 9ProlongedApoptosis requires ATP levels to be maintained,openingInduction ofwhereas in necrosis ATP levels fall. Transientapoptotic cascadeMPT opening allows some swelling of mitochondriaand cytochrome c release, but on resealing ATPlevels can be restored allowing apoptosis to occur.APOPTOSISNECROSIS