Calcium, mitochondria and reperfusion injury A pore way to die

Andrew Halestrap

Department of Biochemistry and

The Bristol Heart Institute

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 synthesisCell 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 nucleotidesHigh phosphateOxidative stress

Conditions occurring when cells damaged e.g. by toxins or hypoxia

Proposed scheme for the mechanism of pore opening

ATP

ADP

Cytosol

Matrix

Normal Impermeable

State

Adeninenucleotide

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 AActivated 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

Ca2+

Binding increased by oxidative stress and thiol reagents. CyP binding increases

sensitivity to [Ca].

ATP

ADP

ImpermeableState

Cyclophilin D

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

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

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

Inhibition of the permeability transition by CsA and SfA is overcome by high [Ca2+]

0 20 40 60 80

[Ca2+

] M

0

10

20

30

40

50

60

70

Rat

e o

f S

hri

nka

ge

(A5

20.s

- 1 x

104)

Control

1 M SfA

10

20

30

40

50

60

0

70

Ra

te o

f sh

rinka

ge

(A5

20.s

.1

0 )

-14

0 20 40 60 80

[Ca2+] M

Shrinkage

0 20 40 60 80

[Ca2+

] M

0

10

20

30

40

50

60

70

Rat

e o

f S

hri

nka

ge

(A5

20.s

- 1 x

104)

10

20

30

40

50

60

0

70

0 20 40 60 80

[Ca2+] M

Shrinkage

Control1 M SfA

10

20

30

40

50

60

0

70

Ra

te o

f sh

rinka

ge

(A5

20.s

.1

0 )

-1.s

.1

0 )

-14

0 20 40 60 80

[Ca2+] M

Shrinkage

or 1 M CsA

Proposed scheme for the mechanism of pore opening

ATP

ADP

Cytosol

Matrix

Normal Impermeable

State

Adeninenucleotide

translocase

Ca

Very high [Ca2+]

Cyclosporin AActivated 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

Ca2+

Binding increased by oxidative stress and thiol reagents. CyP binding increases

sensitivity to [Ca].

ATP

ADP

ImpermeableState

Cyclophilin D

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 (K0.5 4-7nM)

0 5 10 15 20 25 100

[Sanglfehrin] nM

0

1

2

3

4

5

6

Ra

t e c

on

st a

nt

for

pe

pt i

de

hy

dro

lys

is (

s-1

)

[Sanglifehrin] (nM)

PPiase activity of CyP-D

0

1Rat

e co

nst

ant

for

pe

ptid

e h

ydro

lysi

s (s

)-1

2

3

4

5

6

0 5 10 15 20 25 100

Ki 2nM

SfA inhibits the peptidyl prolyl cis trans isomerase activity of CyP-D and inhibits the permeability transition

30 s

A520

0.05

Control

150 nM SfA

150 nM CsA

500 nM SfA

1 MSfA1 M CsA

500 nM CsA

Ca2+ De-energised swelling

Anti-ANT antibody immunoprecipitates CyP-D – inhibited by CsA but not SfA

Cont SfA CsA

20kDa

Proposed scheme for the mechanism of pore opening

ATP

ADP

Cytosol

Matrix

Normal Impermeable

State

Adeninenucleotide

translocase

Ca

Very high [Ca2+]

Cyclosporin AActivated 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

Ca2+

Binding increased by oxidative stress and thiol reagents. CyP binding increases

sensitivity to [Ca].

ATP

ADP

ImpermeableState

Cyclophilin D

Sanglifehrin ASfA

X

Evidence for the involvement of the Adenine Nucleotide Translocase

0 50 100 150 200 250 300 350 400

[Ca2+

] M

0

5

10

15

20

Rat

e o

f S

hri

nka

ge

(A5

20.s

- 1x1

000)

Control

20M ADP

100M ADP

Inhibition of the MPT by ADP

5 150

[Ca2+

] M

0

50

100

150

200

250

Rat

e o

f sh

rin

kag

e (

A5

20.s

-1.1

04)

50 M ADP

50 M ADP +10 M CAT

Control ( ) or10 M BKA ( )

0 25 50 75 100 12

Matrix GSH GST CyP-D

Add Triton-X100-solubilised innermitochondrial membranes

Wash off non-specifically bound protein

GSH

SDS-PAGE and Western blottingwith anti ANT antibodies

Cyclophilin affinity column

Specifically boundprotein

ANT

Western blots

No IMM

CsA + IMM

CsH + IMM

Plus IMM

Porin

Glutathione eluted proteins

30kDa

Control Diamide

More ANT from diamide-treated mitochondria binds GST-CyP-D column

The ANT binds to immobilised CyP-D in a CsA sensitive manner

Inhibition of the MPT by ADP is antagonised by thiol modification

0 200 400 600 800 1000

[ADP]M

0

20

40

60

80

100R

ate

of

sh

rin

kin

g(%

rate

at

zero

AD

P)

Control K i 1.7 M

TBH-treated K i 67 M

PheArs-treated K i 491 M

0 200 400 600 800 1000

[ADP]M

0

20

40

60

80

100R

ate

of

sh

rin

kin

g(%

rate

at

zero

AD

P)

Control K i 1.7 M

TBH-treated K i 67 M

PheArs-treated K i 491 M

Pro61 Cys56

Cys159

ADPbinding

Cys256

Location in the ANT of residues with potentialregulatory significance for the MPT

CyP binding?

LowN-ethylmaleimide

Eosine-maleimide

and high NEM

Eosine maleimide (to block Cys159) blocks ATP inhibition of the MPTP

0 200 400 600 800 1000

[AD P ] M

0

20

40

60

80

100R

ate

of

sh

rin

ka

ge

(A

52

0.s

-1.1

04) 80M E osinemale imide

500M NE M (150M Ca2+

)

50M N E M

(50M Ca2+

)

Control(50M Ca

2+)

Cys159

Cys56

Cys56 plus some Cys159

Pro61 Cys56

Cys159

ADPbinding

Cys256

Location in the ANT of residues with potentialregulatory significance for the MPT

CyP binding?

N-ethyl-maleimide

Eosine-maleimide

Diamide and Phenylarsine oxide cross links Cys159 to Cys256

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-PEYROULA1, CÉCILE DAHOUT-GONZALEZ2, RICHARD KAHN1, VÉRONIQUE TRÉZÉGUET3, GUY J.-M. LAUQUIN3 & GÉRARD BRANDOLIN2

Nature 427, 461 - 465 (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

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?

2 min

A340

0.01

120 M Ca

2 g/ml CyP-D

2+

Data of Jeremy Gillespie

CyP-D

CyP-D

CyP-D + CsA7.5

28 62

250 M Ca7.5

2862

250

6.5

28

62

A340

0.02

3 min

CsA present in buffer

CsA not present in

buffer

M Ca

NADH OAA

MDH MDH

NADHOAA

NADHOAA

NAD+MAL

NAD+MAL

X

A340

CyP-DCa2+

Pore opens

ANT MPTANT

Porin

Silver stain

HA HiT

Does MPTP opening occur in 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)

Long period of ischaemia

Severe damage exacerbatedon reperfusion which leads to

cell death and necrosis

Associated with swollen amorphousmitochondria 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 damageReversible on reperfusion

Reversible reperfusion injury

"Stunned" heart eventually recovers fully

Reperfusion injury

Damage to intracellular components

Repair of damage Cell survives

Further damage Cell dies

MPTP closed - High ATP Low ATP – MPTP open

MitochondriaLIFE DEATHJudge and executioner

ATP depletionDisruption of ionic homeostasis

Calcium overload Oxidative Stress

Ischaemia / Reperfusion

[ 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 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)

ISCHAEMIA0 20 100 50 60 125

3H-DOG-loading

Isolated mitochondria

-1 0 1 2 3 5 15 30

Time (min)

0

10

20

30

40

50

Mit

oc

ho

nd

ria

l 2

-DO

G u

pta

ke

(R

ati

o u

nit

s)

7.1

7.2

7.3

7.4

7.5

7.6

pH

of h

ea

rt efflu

en

t

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

Does prevention of MPTP opening protect hearts from

reperfusion injury?

Protecting hearts from reperfusion injury

Low pH and [Ca2+]mito(Na+/H+ antiporter inhibitors)

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

Cyclosporin A and Sanglifehrin A protects hearts from reperfusion injury

0

20

40

60

80

100

Re

co

ve

ry o

f L

VD

P (

% p

re-i

ec

ha

em

ic v

alu

e)

0

20

40

60

80

100

LV

ED

P (m

m H

g)

LVDP LVEDP

Con SfA CsA Con SfA CsA

80

60

40

100

0

20

80

60

40

100

0

20

LV

DP

(%

pre

isch

aem

ic v

alu

e)

LE

VD

P (m

m H

g)

Data of Sam Clarke and Gavin McStay

Control

SfA

CsA

30 40 80 85 90 95 100 105 110

30 m

in Is

chae

mia

60

50

40

30

10

0

20

Time (min)

*

*

*

*

* *

LDH release

LD

H r

elea

sed

(m

un

its/

ml p

erfu

sate

)

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.

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 insulinHypoglycaemia

30 min + CsAHypoglycaemia

Control 30 min insulinHypoglycaemia

30 min + CsAHypoglycaemia

Toluidine bluestained

Sensitivity of MPT to [Ca] in mitochondria from different regions of the brain

1min

A520

100 M Ca 2mM EGTA

Hippocampus

Cerebellum

Cerebellum

Cortex

Hippocampus

[Ca2+] M

1005020

Rat

e of

sw

ellin

g50

100

150

250

200

Friberg, H.; Connern, C.; Halestrap, A. P., and Wieloch, T. (1999) J. Neurochem. 72: 2488-2497.

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

Ischaemic Preconditioning

Brief ischaemic episodes followed by recovery protect the heart against subsequent prolonged

ischaemia and reperfusion.

Does this involve inhibition of pore opening?

Protection of hearts from reperfusion injury by ischemic preconditioning

** p<0.01

Data of Sabzali Javadov and Kelvin Lim

Ischaemic preconditioning inhibits mitochondrial pore opening

0 2 4 6 8 10 12

0

20

40

60

80

Mito

cho

nd

rial

DO

G u

pta

ke (

Rat

io u

nits

)

Pre-ischemic Post-ischemic Pre-ischemic Post-ischemic

Pre-ischemic DOG loading

Pre-ischemic DOG loading

Control

Preconditioned

DOG pre-loaded

DOG post-loaded**

**

0 15 100 45 70 1303H-DOG-loading 5 5

I I

t (min)

ISCHAEMIA0 95 40 70 125

I 5 5

203H-DOG-loading

135I

ISCHAEMIA

0

1

2

3

4

5

6

7A

52

0 .

103

Control

IP

Pre-ischaemia End of Ischemia 3 min Reperfusion

**

**

** P<0.01 vs. Control

Ischaemic preconditioning decreases Ca2+-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 Ca2+

Data of Igor Khaliulin, Sam Clarke and Jo Parker

0

10

20

30

40

Mit

oc

ho

nd

ria

l D

OG

up

tak

e (

Ra

tio

un

its

)

(4)

(6)

Noischemia

(4)

Pre-ischemicDOG 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-ischemicDOG loading

57.2± 10.3

98.9± 10.8

0

ISCHAEMIA0 100 50 60 125

3H-DOG-loading

+/- 10 mM pyruvate

155 165

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

Effects of PROPOFOL on mitochondrial pore opening and functional recovery during reperfusion of hearts after 30 min ischaemia

LVDP EDP

Control

0

10

20

30

40

50

60

70

LV

DP

an

d E

DP

(m

m H

g)

Propofol

Data of Sabzali Javadov

0

5

10

15

20

25

MP

TP

op

enin

g in

sit

u (

DO

G u

nit

s)

0

20

40

60

80

Rate o

f mito

cho

nd

rial swellin

g at 100 M

Ca

2+

DOG Swelling

Ra

te o

f mito

ch

on

dria

l sw

ellin

g a

t 10

0

M C

a2+

0

0

50

100

150

200

250

300

350

400

450

End XC 20 min post XCBaseline

Myo

card

ial l

act

ate

(% o

f b

ase

line

)

Propofol+Pyruvate

Propofol

Control

Propofol+Pyruvate

Propofol

Control

*

*

Lactate content

0

20

40

60

80

100

120

End XC 20 min post XCBaseline

Myo

card

ial A

TP

(% o

f b

ase

line

)

Propofol+Pyruvate

Propofol

Control

Propofol+Pyruvate

Propofol

Control*

p < 0.05

ATP content

0

5

10

15

20

25

30

35

Ser

um T

ropo

nin

I (n

g/m

l)#

*

†

#

Baseline End XC 5 20 60 120

minutes post XC

Propofol+Pyruvate

Propofol

Control

Propofol+Pyruvate

Propofol

Control

10

15

20

25

30

35

40

45

50

55

60

LV S

trok

e W

ork

(g.m

.m -2

.bea

t -1

)

Baseline 60 minpost XC

120 minpost XC

*

Propofol+Pyruvate

Propofol

Control

Propofol+Pyruvate

Propofol

Control

Troponin I release

Work per beat

Apaf-1

Cyt-c

pro-Caspase-9

dATP

AIFSmacIMM

OMM

Mitochondrion

The role of mitochondria in initiating the apoptotic cascade

t-Bid

Bcl-2

Apoptoticsignal

Activation ofApaf-1

Nuclear effects

Bax

+-

MPTP

-

Caspase inhibitor proteins

-

Activation ofapoptoticcascade

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?

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 openATP is depleted

Severe insultpores remain open

APOPTOSIS

MPT pores closeATP production maintained

Moderate insultpores reseal

The extent of the permeability transition may determine whether cell death is

necrotic or apoptotic following ischaemia and reperfusion

?

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 mitochondriaand 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

Co-workersDirki Balaska

Samantha ClarkeElinor Griffiths

Sabzali JavadovIgor KhaliulinAnna LeungJo Parker

CollaboratorsSaadeh Suleiman,

Gianni AngeliniTadeusz Wieloch

Hans Friberg

Cathal ConnernJeremy Gillespie

Paul KerrSabzali Javadov

Kelvin LimGavin McStay

Kuei Woodfield

Past workers

MRC BBSRC

Wellcome Trust