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

2. X ATP synthesis Cell lives Cell dies ATP breakdown
The Mitochondrial Permeability Transition
A calcium induced non-specific pore
Cyclosporin A X
All solutes < 1500daltons
Mitochondrial inner
membrane
Very selective permeability essential for ATP synthesis
Mitochondria become leaky, swollen and uncoupled
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.
Triggered by high matrix [Ca ] 2+
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
Normal Impermeable State Ca
Triggered by low [Ca 2+]
Pathological Non-specific Pore
Ca2+
Cytosol
ADP
Adenine
nucleotide
translocase
ATP
Matrix
Binding increased by oxidative stress and thiol reagents. CyP binding increases sensitivity to [Ca].
ATP
ADP
Impermeable
State
Cyclophilin D
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)
Cyclosporin A
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

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 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. 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+] 2 4 6 8 [ C a + ] m M 1 3 5 7 R t e o f S h r i n k g ( A . s - x )
Control
SfA 10 20 30 40 50 60 70
Rate of shrinkage (A
520
.s ) 80
[Ca 2+
Shrinkage
1 mM SfA
or 1 mM CsA

8. Proposed scheme for the mechanism of pore opening
Normal Impermeable State Ca
Triggered by low [Ca 2+]
Pathological Non-specific Pore
Ca2+
Cytosol Ca
Very high [Ca2+]
ADP
Adenine
nucleotide
translocase
ATP
Matrix
Binding increased by oxidative stress and thiol reagents. CyP binding increases sensitivity to [Ca].
ATP
ADP
Impermeable
State
Cyclophilin D
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)
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.
CLICK
Cyclosporin A
Sanglifehrin A
Like 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 ) 6 1 6 - s (
30 s
A520
0.05
Control
150 nM SfA
150 nM CsA
500 nM SfA
1 mM SfA
1 mM CsA
500 nM CsA
Ca2+
De-energised swelling s -1 i s 5
PPiase activity of CyP-D y 5 l o r d y 4 h 4
peptide hydrolysis (s )
Rate constant for e d t i 3 p 3 e
Ki 2nM p r o f 2 2 t n a t s 1 n 1 o c e t a R 5 10 15 20 25
100 5 1 1 5 2 2 5 1
[Sanglifehrin] (nM) [ S a n g l f e h r i n ] n M

12. Proposed scheme for the mechanism of pore opening
Normal Impermeable State Ca
Triggered by low [Ca 2+]
Pathological Non-specific Pore
Ca2+
Cytosol Ca
Very high [Ca2+]
ADP
Adenine
nucleotide
translocase
ATP
Matrix
Binding increased by oxidative stress and thiol reagents. CyP binding increases sensitivity to [Ca].
ATP
ADP
Impermeable
State
Cyclophilin D
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)
Sanglifehrin A
SfA X
Cyclosporin A

13. Evidence for the involvement of the Adenine Nucleotide Translocase

14. Inhibition of the MPT by ADP5 1 [ C a 2 + ] m M R t e o f s h r i n k g ( A . - 4 ) D P T l B K 7 5 1 2 3 4 [ C a + ] m M R t e o f S h r i n k g ( A . s - x ) l D P

15. The ANT binds to immobilised CyP-D in a CsA sensitive manner
Western blots
No IMM
CsA + IMM
CsH + IMM
Plus IMM
Porin
Glutathione eluted proteins
Cyclophilin affinity column
Matrix
GSH
GST
CyP-D
Add Triton-X100-solubilised inner
mitochondrial membranes
Wash off non-specifically bound protein
GSH
Specifically bound
protein
30kDa
Control
Diamide
More ANT from diamide-treated mitochondria binds GST-CyP-D column
SDS-PAGE and Western blotting
with 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 MPT
Pro
ADP
Cys 61
Low
N-ethylmaleimide 56
binding
Cys
Eosine-
Cys
159
256
maleimide and high NEM
CyP
binding?

18. Eosine maleimide (to block Cys159) blocks ATP inhibition of the MPTP2 4 6 8 1 [ A D P ] m M R a t e o f s h r i n k g ( 5 . - ) E l d N C +
Cys159
Cys56
Cys56 plus some Cys159

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

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 carboxyatractyloside
EVA 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 pore
JASON 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. WALLACE1,3,4
Key point:
Mice with a double ANT knockout in their liver mitochondria still show a cyclosporin-sensitive MPTP. However, it is less sensitive to 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
340
OAA
MAL X
ANT
Porin
Silver stain HA
HiT
CyP-D
ANT
MPT Ca 2+
NADH
NAD+
OAA
MAL
MDH
Pore opens
MDH
CyP-D
CyP-D + CsA
7.5 28 62
250 m
M Ca
6.5 A
340
0.02
3 min
CsA present
in buffer
CsA
not
present in
buffer
2 min A
340
0.01
120 m
M Ca 2
g/ml CyP-D 2+
Data of Jeremy Gillespie

24. Does MPTP opening occur in reperfusion injury?

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 ionic
disturbances (e.g. lactic acid build up, low pH, high [Na] and [Ca], free radicals)
Short period of ischaemia
Mild to moderate damage
Reversible on reperfusion
Reversible reperfusion injury
"Stunned" heart eventually
recovers fully
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

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

27. The "Hot DOG" technique for measuring pore opening
[ H]-DOG (2-deoxyglucose) 3
Load heart cells with 2-deoxyglucose
(DOG) which is trapped in the cytosol as DOG-6-P
DOG
DOG-6-P
DOG-6-P X
Pore closed
Pore open
DOG-6-P only enters mitochondria if pore opens
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 20
100 50 60
125
3H-DOG-loading
Isolated 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 ischaemia pH
DOG
Pore opens as pH returns to normal. (Pore is inhibited at pH<7.0)
30 min ischemia PI
Time of postischaemic reperfusion (min)
- 0
- 100
mm Hg
Ischaemia
Data 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 MPTP
Pyruvate
Less oxidative stress
(Free radical scavengers e.g. propofol)
Low pH and [Ca2+]mito
Ischaemic Preconditioning

31. Cyclosporin A and Sanglifehrin A protects hearts from reperfusion injury
Data of Sam Clarke and Gavin McStay
LVDP
LVEDP
Con
SfA
CsA 80 60 40
100 20
LVDP (% preischaemic value)
LEVDP (mm Hg)
Control
SfA
CsA 30 40 80 85 90 95
100
105
110
30 min Ischaemia 60 50 10 20
Time (min) *
LDH release
LDH released (munits/ml perfusate)

32. Baines et al (2005). Nature, 434: 658-662.
The hearts of CyP-D knockout mice are protected from reperfusion injury
Baines 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 blue
stained
Control
30 min insulin
Hypoglycaemia
30 min + CsA
Hypoglycaemia
Control
30 min insulin
Hypoglycaemia
30 min + CsA

34. Sensitivity of MPT to [Ca] in mitochondria from different regions of the brain
Cerebellum
Cortex
Hippocampus
[Ca2+] mM
100 50 20
Rate of swelling
150
250
200
100 mM Ca
2mM EGTA
Cerebellum
A520
Hippocampus
1min
Friberg, H.; Connern, C.; Halestrap, A. P., and Wieloch, T. (1999) J. Neurochem. 72:

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

36. Does this involve inhibition of pore opening?
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. Data of Sabzali Javadov and Kelvin Lim
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 ** ** 15 45 70
100
130
3H-DOG-loading I I
ISCHAEMIA 5 5
ISCHAEMIA 95 40 70
125 I 5 20
3H-DOG-loading
135
t (min)

39. Pre-ischaemia End of Ischemia 3 min Reperfusion
Ischaemic preconditioning decreases Ca2+-dependent swelling of mitochondria isolated following Ischaemia/Reperfusion 7
Control 6 IP 5
** P<0.01
vs. Control A 4 ** 3 2 ** 1
Pre-ischaemia End of Ischemia 3 min Reperfusion
Rapidly 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 ischemia
Pyruvate-treated ischemia
36.2
± 9.9
Control 40
LVDP
% control
(6)
10 mM Pyruvate
105.3
± 17.2
(5)
(4)
Post-ischemic
DOG loading
57.2
± 10.3
98.9
± 10.8
ISCHAEMIA
100 50 60
125
3H-DOG-loading
+/- 10 mM pyruvate
155
165
t (min)
Post-ischaemic loading
Pyruvate is:
a free radical scavenger
a good respiratory substrate for ATP production
It causes acidification 30
(4)
Mitochondrial DOG uptake (Ratio units) 20
Pre-ischaemic loading
(4)
+/- 10 mM pyruvate 20 50 60
100
125 10
3H-DOG-loading
ISCHAEMIA
t (min) No
Pre-ischemic
ischemia
DOG loading
Data of Paul Kerr

41. Effects of PROPOFOL on mitochondrial pore opening and functional recovery during reperfusion of hearts after 30 min ischaemia
Control
Propofol 5 10 15 20 25
MPTP opening in situ (DOG units) 40 60 80
Rate of mitochondrial swelling at 100 m
M Ca 2+
DOG
Swelling
Rate of mitochondrial swelling at 100 mM Ca2+
LVDP
EDP 70 60 50
LVDP and EDP (mm Hg) 40 30 20 10
Data of Sabzali Javadov

43. Lactate content
ATP content

44. Troponin I release
Work per beat

45. The role of mitochondria in initiating the apoptotic cascade
t-Bid
Bcl-2
Apoptotic
signal
Activation of
Apaf-1
Nuclear
effects
Bax + -
MPTP
Apaf-1
Cyt-c
pro-Caspase-9
dATP
AIF
Smac
IMM
OMM
Mitochondrion
Activation of
apoptotic
cascade
cleavage
Caspase-9
pro-Caspase-3
Caspase-3
Proteolytic -
Caspase
inhibitor
proteins
MPTP 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 Transition
Mitochondrial swelling and outer membrane rupture
Release of cytochrome c, AIF and Smac/Diablo
Activation of caspases
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
NECROSIS
MPT pores stay open
ATP is depleted
Severe insult
pores remain open

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

48. MRC
BBSRC
Wellcome Trust