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