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Calcium, mitochondria and reperfusion injury A pore way to die Andrew Halestrap Department of Biochemistry and The Bristol Heart Institute.

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Presentation on theme: "Calcium, mitochondria and reperfusion injury A pore way to die Andrew Halestrap Department of Biochemistry and The Bristol Heart Institute."— Presentation transcript:

1 Calcium, mitochondria and reperfusion injury A pore way to die Andrew Halestrap Department of Biochemistry and The Bristol Heart Institute

2 The Mitochondrial Permeability Transition A calcium induced non-specific pore All solutes < 1500daltons Mitochondria become leaky, swollen and uncoupled Triggered by high matrix [Ca ] Mitochondrial inner membrane 2+ Very selective permeability essential for ATP synthesis Cyclosporin A X ATP synthesis Cell lives Cell dies ATP breakdown If the pore opens, not only are mitochondria unable to make ATP, they also breakdown ATP made by functional mitochondria and glycolysis. Sensitised to Ca by: Low adenine nucleotides High phosphate Oxidative stress Conditions occurring when cells damaged e.g. by toxins or hypoxia

3 Proposed scheme for the mechanism of pore opening ATP ADP Cytosol Matrix Normal Impermeable State Adenine nucleotide translocase Note that outer membrane proteins such as VDAC (porin), Bcl2 family members and the peripheral benzodiazipine receptor may be involved as regulatory or structural components Cyclosporin A Activated by thiol reagents and oxidative stress which decrease ADP/ATP binding Inhibited by [Mg 2+ ], low pH, adenine nucleotides and membrane potential (which increases ATP binding) Ca Triggered by low [Ca 2+ ] Pathological Non-specific Pore Ca 2+ Binding increased by oxidative stress and thiol reagents. CyP binding increases sensitivity to [Ca]. ATP ADP Impermeable State Cyclophilin D

4 Evidence for the involvement of Cyclophilin D For all CsA analogues tested the K 0.5 for inhibition of the peptidyl-prolyl cis-trans isomerase activity of CyP-D correlates with the K 0.5 for inhibition of the MPTP The 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 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: 658-662. 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: 18558-18561. 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: 652-658. 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: 12005-12010. CyP-D knockout mice show impaired permeability transition

6 The MPTP opens in CyP-D knockout mitochondria at high calcium loading

7 Inhibition of the permeability transition by CsA and SfA is overcome by high [Ca 2+ ] 020406080 [Ca 2+ ] M 0 10 20 30 40 50 60 70 R a t e o f S h r i n k a g e ( A 5 2 0. s - 1 x 1 0 4 ) Control 1 MSfA 10 20 30 40 50 60 0 70 Rate of shrinkage (A 520.s.10 ) - 1 4 0 20 406080 [Ca 2+ ] M Shrinkage 020406080 [Ca 2+ ] M 0 10 20 30 40 50 60 70 R a t e o f S h r i n k a g e ( A 5 2 0. s - 1 x 1 0 4 ) 10 20 30 40 50 60 0 70 0 20 406080 [Ca 2+ ] M Shrinkage Control 1 M SfA 10 20 30 40 50 60 0 70 Rate of shrinkage (A 520.s.10 ) - 1 - 1 4 0 20 406080 [Ca 2+ ] M Shrinkage or 1 M CsA

8 Proposed scheme for the mechanism of pore opening ATP ADP Cytosol Matrix Normal Impermeable State Adenine nucleotide translocase Ca Very high [Ca 2+ ] Cyclosporin A Activated by thiol reagents and oxidative stress which decrease ADP/ATP binding Inhibited by [Mg 2+ ], low pH, adenine nucleotides and membrane potential (which increases ATP binding) Ca Triggered by low [Ca 2+ ] Pathological Non-specific Pore Ca 2+ Binding increased by oxidative stress and thiol reagents. CyP binding increases sensitivity to [Ca]. ATP ADP Impermeable State Cyclophilin D

9 The Immunosuppressive drug Sanglifehrin A is a novel inhibitor of the MPTP Cyclosporin A Sanglifehrin A Unlike CsA, the CyPA-SfA complex has no effect on calcineurin Like CsA, SfA also binds tightly to CyP- A (K 0.5 4-7nM)

10 0510152025100 [Sanglfehrin] nM 0 1 2 3 4 5 6 R a t e c o n s t a n t f o r p e p t i d e h y d r o l y s i s ( s - 1 ) [Sanglifehrin] (nM) PPiase activity of CyP-D 0 1 Rate constant for peptide hydrolysis (s ) 2 3 4 5 6 051015 20 25100 Ki 2nM SfA inhibits the peptidyl prolyl cis trans isomerase activity of CyP-D and inhibits the permeability transition 30 s A 520 0.05 Control 150 nM SfA 150 nM CsA 500 nM SfA 1 M SfA 1 M CsA 500 nM CsA Ca 2+ De-energised swelling

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12 Proposed scheme for the mechanism of pore opening ATP ADP Cytosol Matrix Normal Impermeable State Adenine nucleotide translocase Ca Very high [Ca 2+ ] Cyclosporin A Activated by thiol reagents and oxidative stress which decrease ADP/ATP binding Inhibited by [Mg 2+ ], low pH, adenine nucleotides and membrane potential (which increases ATP binding) Ca Triggered by low [Ca 2+ ] Pathological Non-specific Pore Ca 2+ Binding increased by oxidative stress and thiol reagents. CyP binding increases sensitivity to [Ca]. ATP ADP Impermeable State Cyclophilin D Sanglifehrin ASfA X

13 Evidence for the involvement of the Adenine Nucleotide Translocase

14 Inhibition of the MPT by ADP 5150 [Ca 2+ ] M 0 50 100 150 200 250 R a t e o f s h r i n k a g e ( A 5 2 0. s - 1. 1 0 4 ) 50 M ADP 50 M ADP + 10 M CAT Control () or 10 M BKA () 025507510012

15 MatrixGSHGSTCyP-D Add Triton-X100-solubilised inner mitochondrial membranes Wash off non-specifically bound protein GSH SDS-PAGE and Western blotting with anti ANT antibodies Cyclophilin affinity column Specifically bound protein 30kDa ControlDiamide More ANT from diamide-treated mitochondria binds GST-CyP-D column The ANT binds to immobilised CyP-D in a CsA sensitive manner

16 Inhibition of the MPT by ADP is antagonised by thiol modification

17 Pro 61 Cys 56 Cys 159 ADP binding Cys 256 Location in the ANT of residues with potential regulatory significance for the MPT CyP binding? Low N-ethylmaleimide Eosine- maleimide and high NEM

18 Eosine maleimide (to block Cys 159 ) blocks ATP inhibition of the MPTP

19 Pro 61 Cys 56 Cys 159 ADP binding Cys 256 Location in the ANT of residues with potential regulatory significance for the MPT CyP binding? N-ethyl- maleimide Eosine- maleimide Diamide and Phenylarsine oxide cross links Cys 159 to Cys 256

20 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, 39 - 44 (06 November 2003) Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside EVA PEBAY-PEYROULA 1, CÉCILE DAHOUT-GONZALEZ 2, RICHARD KAHN 1, VÉRONIQUE TRÉZÉGUET 3, GUY J.-M. LAUQUIN 3 & GÉRARD BRANDOLIN 2

21 Nature 427, 461 - 465 (29 January 2004); The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore JASON E. KOKOSZKA 1,4,*, KATRINA G. WAYMIRE 1,4, SHAWN E. LEVY 4,*, JAMES E. SLIGH 4,*, JIYANG CAI 5, DEAN P. JONES 5, GRANT R. MACGREGOR 1,2,4 & DOUGLAS C. WALLACE 1,3,4 Key point: Mice with a double ANT knockout in their liver mitochondria still show a cyclosporin-sensitive MPTP. However, it is less sensitive to Ca 2+ 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 2 min A 340 0.01 120 M Ca 2 g/ml CyP-D 2+ Data of Jeremy Gillespie CyP-D CyP-D + CsA 7.5 28 62 250 M Ca 7.5 28 62 250 6.5 28 62 A 340 0.02 3 min CsA present in buffer CsAnot present in buffer M Ca NADH OAA MDH NADH OAA NADH OAA NAD+ MAL NAD+ MAL X A 340 CyP-D Ca 2+ Pore opens ANT MPT ANT Porin Silver stain HAHiT

24 Does MPTP opening occur in reperfusion injury?

25 Ischaemia (major reduction or total loss of blood flow) ATP drops and cell begins to deteriorate as a result of metabolic and ionic disturbances (e.g. lactic acid build up, low pH, high [Na] and [Ca], free radicals) Long period of ischaemia Severe damage exacerbated on reperfusion which leads to cell death and necrosis Associated with swollen amorphous mitochondria that are uncoupled and have impaired respiration. They look as if the pore has opened. Irreversible reperfusion injury Short period of ischaemia Mild to moderate damage Reversible on reperfusion Reversible reperfusion injury "Stunned" heart eventually recovers fully Reperfusion injury

26 Damage to intracellular components Repair of damage Cell survives Further damage Cell dies MPTP closed - High ATP Low ATP – MPTP open Mitochondria LIFEDEATH Judge and executioner ATP depletion Disruption of ionic homeostasis Calcium overload Oxidative Stress Ischaemia / Reperfusion

27 [ H]-DOG (2-deoxyglucose) 3 DOG DOG-6-P Load heart cells with 2-deoxyglucose (DOG) which is trapped in the cytosol as DOG-6-P DOG-6-P X Pore closed Pore open DOG-6-P only enters mitochondria if pore opens The "Hot DOG" technique for measuring pore opening The 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) ISCHAEMIA 0 20 100 50 60 125 3H-DOG-loading Isolated mitochondria

28 pH DOG 30 min ischemia Time of postischaemic reperfusion (min) PI Time dependence of mitochondrial pore opening and pH recovery during reperfusion of hearts after 30 min ischaemia Pore opens as pH returns to normal. (Pore is inhibited at pH<7.0) Data of Paul Kerr - 0 - 100 mm Hg Ischaemia

29 Does prevention of MPTP opening protect hearts from reperfusion injury?

30 Protecting hearts from reperfusion injury Low pH and [Ca 2+ ] mito Less oxidative stress (Free radical scavengers e.g. propofol) (Cyclosporin A, Sanglifehrin A and CyP-D knockout) Direct inhibitors of the MPTP Pyruvate Ischaemic Preconditioning

31 Cyclosporin A and Sanglifehrin A protects hearts from reperfusion injury LVDP LVEDP ConSfACsAConSfACsA 80 60 40 100 0 20 80 60 40 100 0 20 LVDP (% preischaemic value) LEVDP (mm Hg) Data of Sam Clarke and Gavin McStay

32 The hearts of CyP-D knockout mice are protected from reperfusion injury Baines et al (2005). Nature, 434: 658-662. Nakagawa et al (2005).. Nature, 434: 652-658.

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: 5151-5159. 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: 736-741. Control 30 min insulin Hypoglycaemia 30 min + CsA Hypoglycaemia Control 30 min insulin Hypoglycaemia 30 min + CsA Hypoglycaemia Toluidine blue stained

34 Sensitivity of MPT to [Ca] in mitochondria from different regions of the brain 1min A 520 100 M Ca 2mM EGTA Hippocampus Cerebellum Cortex Hippocampus [Ca 2+ ] M 10050 20 Rate of swelling 50 100 150 250 200 Friberg, H.; Connern, C.; Halestrap, A. P., and Wieloch, T. (1999) J. Neurochem. 72: 2488-2497.

35 Copyright ©2005 by the National Academy of Sciences Schinzel, Anna C. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 12005-12010 Ischemia/reperfusion-mediated brain damage in WT and CypD- deficient mice

36 Ischaemic Preconditioning Brief ischaemic episodes followed by recovery protect the heart against subsequent prolonged ischaemia and reperfusion. Does this involve inhibition of pore opening?

37 Protection of hearts from reperfusion injury by ischemic preconditioning ** p<0.01 Data of Sabzali Javadov and Kelvin Lim

38 Ischaemic preconditioning inhibits mitochondrial pore opening DOG pre-loaded DOG post-loaded ** 0 15 100 45 70 130 3H-DOG- loading 5 5 II t (min) ISCHAEMIA 0 95 40 70 125 I 5 5 20 3H-DOG- loading 135 I ISCHAEMIA

39 0 1 2 3 4 5 6 7 A 520.10 3 Control IP Pre-ischaemia End of Ischemia 3 min Reperfusion ** ** P<0.01 vs. Control Ischaemic preconditioning decreases Ca 2+ -dependent swelling of mitochondria isolated following Ischaemia/Reperfusion Rapidly isolated mitochondria incubated under de-energised conditions in the presence of a calcium ionophore with swelling initiated by addition of 50 M Ca 2+ Data of Igor Khaliulin, Sam Clarke and Jo Parker

40 0 10 20 30 40 Mitochondrial DOG uptake (Ratio units) (4) (6) No ischemia (4) Pre-ischemic DOG loading Pyruvate-treated ischemia Control ischemia LVDP % control 36.2 ± 9.9 105.3 ± 17.2 t (min) ISCHAEMIA 20 100 50 60 125 +/- 10 mM pyruvate Pyruvate protects hearts from reperfusion injury Control 10 mM Pyruvate Pre-ischaemic loading (5) (4) Post-ischemic DOG loading 57.2 ± 10.3 98.9 ± 10.8 0 ISCHAEMIA 0 100 50 60 125 3H-DOG-loading +/- 10 mM pyruvate 155165 t (min) Post-ischaemic loading 3H-DOG-loading Pyruvate is: a free radical scavenger a good respiratory substrate for ATP production It causes acidification Data of Paul Kerr

41 Effects of PROPOFOL on mitochondrial pore opening and functional recovery during reperfusion of hearts after 30 min ischaemia LVDPEDP Control 0 10 20 30 40 50 60 70 LVDP and EDP (mm Hg) Propofol Data of Sabzali Javadov

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43 Lactate content ATP content

44 Troponin I release Work per beat

45 Apaf-1 Cyt-c pro-Caspase-9 dATP AIF Smac IMM OMM Mitochondrion The role of mitochondria in initiating the apoptotic cascade - Caspase inhibitor proteins - Activation of apoptotic cascade cleavage Caspase-9 pro-Caspase-3 Caspase-3 Proteolytic MPTP opening causes swelling and release of intermembrane proteins. Could MPTP opening be involved in apoptosis?

46 Stress to cell Activation of caspases Mitochondrial Permeability Transition Release of cytochrome c, AIF and Smac/Diablo bcl-2 - ? Mitochondrial swelling and outer membrane rupture NECROSIS MPT pores stay open ATP is depleted Severe insult pores remain open APOPTOSIS MPT pores close ATP production maintained Moderate insult pores reseal The extent of the permeability transition may determine whether cell death is necrotic or apoptotic following ischaemia and reperfusion

47 ? Stresses e.g. reperfusion or toxins Induction of apoptotic cascade APOPTOSIS Mitochondrial Permeability Transition Release of cytochrome c, Smac/Diablo and AIF NECROSIS Prolonged opening Role of the Mitochondrial Permeability Transition in Apoptosis and Necrosis Transient opening Apoptosis requires ATP levels to be maintained, whereas in necrosis ATP levels fall. Transient MPT opening allows some swelling of mitochondria and cytochrome c release, but on resealing ATP levels can be restored allowing apoptosis to occur. bcl-2 - Activation of caspase 3 Activation of caspase 9 Swelling and outer membrane rupture bcl-2 - Removal of growth factors Cytokines e.g. TNF, Fas Activation of Caspase 8 ? t-Bid and Bax migrate to mitochondrial outer membrane Multiple and interacting signalling pathways

48 MRCBBSRC Wellcome Trust


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