Mitochondria in ischemia and reperfusion Fabio Di Lisa Department of Biomedical Sciences University of Padova [email protected] CBCS Summer School on Cardiovascular Sciences Basic Mechanisms translated to the Clinic EHH, June 16-20, 2013
Mitochondria in ischemia and reperfusion
Fabio Di Lisa
Department of Biomedical SciencesUniversity of [email protected]
CBCS Summer School on Cardiovascular SciencesBasic Mechanisms translated to the Clinic
EHH, June 16-20, 2013
Mitochondria in ischemia and reperfusion
• (boring) background• Ca2+ and permeability transition• ROS formation
CBCS Summer School on Cardiovascular SciencesBasic Mechanisms translated to the Clinic
EHH, June 16-20, 2013
ETC
FoF1
inner mitochondrial membrane
H+
H+
e- O2
ADP+Pi ATP
H+
ADP+Pi ATP
ETC
FoF1e- O2
inner mitochondrial membranematrix
ox stress
matrix
oligomycin
MITOCHONDRIA AND MYOCARDIAL ISCHEMIA
• Structural and functional abnormalities of mitochondria are caused by ischemia/reperfusion
• Mitochondrial dysfunction might result in myocardialprotection
60 min ischemia40 min ischemia +20 min reperfusion
AC Shen and RB Jennings, Am.J.Pathol., 67:417-440, 1972
Calcium accumulatiom within mitochondrial matrix of
cardiomyocytes induced by post-ischemic reperfusion in dog hearts
0
2
4
6C
Kr e
l ea
se
(IU
/mi n
/ gw
w)
0 60 120 180 240Time (minutes)
anoxia reoxygenation
control
KCN
redrawn from Ganote et al., 1976
lack ofcontraction
rigorcontracture
hypercontractureand loss of
viabilility
5 min
ischemia reperfusionnormoxia
ATP depletion
Suboptimal recovery of mitochondrial function
(low ATP) in the presence of elevated [Ca2+]
lack of oxygen
increased anaerobic glycolysis
inhibition of oxidative phosphorylation
Pi increase
acidosis
decreased ATP synthesis and increased ATP hydrolysis
readmission of oxygen
0
100
LVP
(mm
Hg)
anoxia
reoxygenation
adapted from Siegmund et al., Am. J. Physiol, 1991
reoxygenation
hypercontracture
irreversible loss of
structure and function
recovery of myocyte
morphology and function
slow
(> 5 min)
mitochondrial ATPproduction
partial
Dym recovery
cytosolic ATP+
high [Ca2+]c
( 500nM)
cytosolic ATP+
low [Ca2+]c
(< 200nM)fast
(< 1 min)
60
60
40
20
4 3 25
Rod
cel
ls (%
of t
otal
)
-log [Mg-ATP]
R.A. Altschuld et al., J.Biol. Chem., 260:14325-30, 1985
6.2 6.6 7.05.80
40
20
60
80
pCa
Rod
cel
ls (%
of t
otal
)
53
1.0
0.1
10no ATP
[ATP] mM
Relaxation and maintenance of rod-shaped morphologyrequire [ATP] in the millimolar range
Submillimolar [ATP] results in hypercontracture, a process facilitated by high [Ca2+]
80 60 40 200
10
20
30
80 60 40 200
10
20
30
num
ber o
f cel
ls (%
)
cell length (%)
anoxiareoxygenationreoxy + BDM
ROD
healthy SQUARE
reversibleROUND
irreversible
redrawn from Siegmund et a., Am.J.Physiol., 1991
inhibition of electron transport
ischemia
ATPdepletion
reduced ATP synthesis
increased ATP hydrolysis
increasedROSproduction
oxidative damage of mitochondrial lipids and proteins
release ofpro-apoptoticproteins
impaired Ca2+
homeostasis
PTP openingDym decreaseCell death
The Permeability Transition Pore
The mitochondrial permeability transition (PT) defines a sudden increase in the permeability of the inner mitochondrial membrane to solutes with molecular masses up to 1500 Da.
This process is attributed to the opening of a voltage-and Ca2+-dependent, cyclosporin A (CsA)-sensitive, high-conductance channel that is termed permeability transition pore (PTP)
ANT
VDAC
PBR (TSPO)
CsA CyP-D (Ppif gene)
in press
0 pA
10 pA
500 ms
- 60 mV
0 pA- 40 mV
0 pA10 pA
500 ms
+ 60 mV
0 pA
+ 40 mV
0 pA
0 pA
0 pA
500 ms
500 ms10 pA
10 pA
0 pA
Currents can be elicited by Ca2+ and Pi alone
2 sec
10 pAV
cis: -60 mV
No currents are seen with the monomer
or with gel-purified complex I
Pi
ADP
Mg2+
pH
RNS
Ca2+
ROS
GSH/GSSG
NAD(P)H/NAD(P)+
Soluble factors
HK
PiC
VDAC
ANT
CyPD
GSK3b
PKCe
Protein unfolding
Proteins
Surface potential
DHA
DYm
Arachidonate
IMM functionand lipids
CyPD matrix
inner mitochondrial
membrane
III IVc
I PTP
outer mitochondrial
membrane
intermembrane
space
Ca2+
ROS
ATPase
ROSformation
matrix
inner mitochondrial
membrane
DYm collapse ATP depletion
release ofaccumulated Ca2+
increase in cytosolic Ca2+
NAD+ release NAD+ hydrolysisand depletion
respiratory inhibition
matrixswelling
permeabilization of theouter mitochondrialmembrane
release of cytochrome cand proapoptotic proteins
PTPopening
CyPD matrix
inner mitochondrial
membrane
III IVc
I PTP
outer mitochondrial
membrane
intermembrane
space
Ca2+
ROS
ATPase
CsA
C. Piot, P. Croisille, P. Staat, H. Thibault, G. Rioufol, N. Mewton, R. Elbelghiti, T. Tri Cung, E. Bonnefoy, D.
Angoulvant, C. Macia, F. Raczka, C. Sportouch, G. Gahide, G. Finet, X. André-Fouët, D. Revel, G. Kirkorian, J-P.
Monassier, G. Derumeaux and M. Ovize
N Engl J Med 2008;359:473-81.
Ca2+
DIRECT
matrix
inner mitochondrial
membrane
Spectrophotometric assessments of PTP opening
0 200 400 600 800 1000
0,35
0,40
0,45
0,50
0,55
0,60
0,65
0,70
0,75
Time,s
Ca2+
0 200 400 600 800 1000
0,35
0,40
0,45
0,50
0,55
0,60
0,65
0,70
0,75
Time,s
Abs
orba
nce
540
nm
In isolated mitochondria PTP opening requires the addition of high (i.e., non physiological) Ca2+
Ca2+
Ca2+
DIRECT
INDIRECTPhospholipase A2
Arachidonic Acid CalpainROS
formation
matrix
inner mitochondrial
membrane
0 10 20 30 40 50 60 70
20
30
40
50
60
70
80
90
JC1
fluor
esce
nce
ratio
(597
/539
)
Time (min)
KVAKVA + CsAKVA + CalpeptinaKVA + PD 150606
Mitochondrial dysfunction induced by intracellular Ca2+ overload is decreased by calpain or PTP inhibition
KVA: KCl, vanadate, A23187
0 10 20 30 40 50 600
10
20
30
40
50
60
70
80
90
LDH
rele
ase
(% o
f tot
al)
Time (min)
KVAKVA +CsAKVA +calpeptinKVA +PD
Cell death induced by intracellular Ca2+ overload isdecreased by calpain or PTP inhibition
KVA: KCl, vanadate, A23187
T=0min KV T=10min KV T=30min KV T=60min KV
+ A23
GREEN = Calpastatin-GFP expressing cellsRED = Trypan blue
Does CsA protection depend only on PTP inhibition?
Proteins (2008) 70, 1635-1639
Red: 5 out of 5Orange: 3 out of 4Yellow: 2 out of 4Green: 1 out of 4Blue: unique
Conserved between
Cyp A,B,C,D,E
Does CsA protection depend also on sites other than CypD?
calcineurinactivation
DRP1dephosphorylation
DRP1 translocationto mitochondria
mitochondriafragmentation
[Ca2+]c increase
CsA
mdivi-1
mitochondrialdeenergization
Mitochondrial dynamics
Fusion Fission
OPA1 (IMM)Mfn 1 and 2 (OMM) Drp1
Drp1
b-Actina
Grp75
Opa1
To
t
To
t
To
t
Cit
Cit
Cit
Mit
o
Mit
o
Mit
o
To
t
To
t
To
t
Cit
Cit
Cit
Mit
o
Mit
o
Mit
o
Control Isc/Rip
+MDVIIsc/Rip Isc/RipIsc/Rip
+CsAIsc/Rip
+FK506
Drp1
b-Actina
Grp75
Opa1
WT
Cp
D -
/-Mdivi-1 prevents I/R-induced translocation of DRP-1 to
mitochondria in perfused mouse hearts
0
10
20
30
40
50
P<0.05
P<0.001
P<0.01
P<0.001P<0.05
LDH
rele
ase
(% o
ftot
al)
CsA-induced protection against I/R injury results from addingPTP desensitization with DRP-1 inhibition
calcineurinactivation PTP opening
DRP1dephosphorylation
alteration ofmitochondrial function
DRP1 translocationto mitochondria
mitochondriafragmentation
alteration ofmitochondrial structure
[Ca2+] increase
cell death
CsA
mdivi-1
Cyp-D
Is Cyp-D inhibition/deletion always beneficial?
Advantages
- Decreased susceptibility to PTP opening decreased cell death (necrosis)
Disadvantages
- Increased activity of FoF1 ATPase possible increase in ATP hydrolysis
- Inceased matrix [Ca2+] content deranged substrate oxidation
- Decreased interaction with Bcl2 increased apoptosis
*
100
200
WT KO%
ATP
hyd
roly
sis
1 2Anti-F1
μM CsA0.8 1.60 0.8 1.60
1 2
*
μM CsA
100
200
% A
TP h
ydro
lysi
s
Anti-F1
A B
Anti-CyPD Anti-CyPD
- CyP-D binds to FOF1 ATPase- Diplacement of CyPD by CsA increases ATP synthesis and hydrolysis- Mitochondria lacking CyPD display a higher FOF1 ATPase activity
V. Giorgio et al., JBC 284:33982-8, 2009
Cyp-D KO mice exhibit substantially greater cardiac hypertrophy, fibrosis, and reduction in myocardial function in response to pressure overload and sustained exercise than control mice.
The maladaptive phenotype in the hearts of Cyp-D KO mice was associated with an alteration in PTP-mediated Ca2+ efflux resulting in elevated levels of mitochondrial matrix Ca2+.
PTP appears to maintain homeostatic mitochondrial Ca2+ levels to match metabolism with alterations in myocardial workload.
J. Clin. Invest. 120:3680-7, 2010
CypD interacts with Bcl2 as confirmed with co-immunoprecipitation,pulldown, and mammalian two-hybrid assays.
Cyclosporine A, disrupts the CypD-Bcl2 interaction.
CypD has a limiting effect on cytochrome c release from mitochondria. Such an effect of CypD is cyclosporine A- and Bcl2-dependent.
Overexpression or knockdown of CypD respectively decreases or increases cytochrome c release from mitochondria.
Decreased PTP opening
Increased mitochondrial [Ca2+]
Adverse metabolic remodelling
Increased propensity to failure
Inhibition of CyPD-Bcl2 interaction
Increased occurrence of
apoptosis
Decreased PTP opening
Maintenance of Dym
Decreased ATP and PN hydrolysis
Reduced oxidative stress
and cytochrome crelease
Reduced cell death
The yinyang of PTP inhibition
Cardioprotection afforded by CsA might be contributed by actions at sites other than CypD.
CypD (and PTP) inhibition is likely to be not alwaysbeneficial.
inhibition of electron transport
ischemia
ATPdepletion
reduced ATP synthesis
increased ATP hydrolysis
increasedROSproduction
oxidative damage of mitochondrial lipids and proteins
release ofpro-apoptoticproteins
impaired Ca2+
homeostasis
PTP openingDym decreaseCell death
H2O2-dependent changes in CRC of mouse heart mitochondria
Mutation of cysteine 203 of cyclophilin D inhibits mPTP opening and improves cell viability
i.e., oxidation of Cys203 in CyPD is likely to be
required for PTP opening
M. Giorgio et al., Nat Rev Mol Cell Biol, 8:722-728, 2007
D.L Hoffman, P.S. Brookes, JBC, 284:16236–16245, 2009
16236–16245
Reviewer's argument
Experimental data showing that mitochondria from aged animals produce more ROS should be presented.
(weak) response
Direct evidence is not available. The increased formation
of ROS in mitochondria from aged animals is indirectly
supported by mtDNA oxidation, lipoperoxidation and
increased susceptibility to ischemic damage.
matrix
inner mitochondrial
membrane
III IVcI
PTP
O2-.
O2-.
p66Shc
e-
MAO
serotonin catecholamines
SOD
H2O2SOD
aldehydes NH4+
outer mitochondrial
membrane
intermembrane
space
NOX4
“It is noteworthy that the brain intramitochondrial [H2O2]ss obtainedduring the monoamine oxidase-catalyzed oxidative deamination oftyramine is 48-fold higher than that originating during theoxidation of substrates via complex II of the electron transfer chainin the presence of Antimycin A.”
Cadenas and Davies, Free Radic Biol Med. 2000 Aug;29(3-4):222-30
FAD-binding
domain
Substrate-
binding
domain Membrane
anchor
Overall Structure of Human MAO A
OMM
Advantages with studying MAO and its inhibition
• Molecular structure identified
• Specific substrates
• Clinically available inhibitors
Two isoforms: MAO A and MAO B
In rat heart mitochondria MAO A is the prevailing isoform
Substrates: MAO A, serotonine, norepinephrineMAO B, dopamineMAO and B, tyramine
Inihibitors: MAO A, ClorgylineMAO B, DeprenylMAO A and B, Pargyline
Can MAO activity directly
target mitochondrial function?
MAO activation and mitochondrial function
0
20
40
60
80
100
120
CT DA DA+oligo DA+oligo+parg CT+oligo
#
*
TMR
M fl
uore
scen
ce (v
s co
ntro
l)
N. Kaludrcic et al., ARS 2013
MAO ALDH2DA DOPAL DOPAC
Products of MAO activity and mitochondrial function
#
*
TMR
M fl
uore
scen
ce (v
s co
ntro
l)
cyanamide
0
20
40
60
80
100
120
CT DA DA parg
scramble siRNA
Mitochondrial membrane potential
ALDH2
GAPDH
siRNA 24h siRNA 48hscramble
0
0,2
0,4
0,6
0,8
1
1,2
scramble 24h 48h
ALDH2/gapdh
siRNA
Aldehyde generation and mitochondrial function
MAO ALDH2DA DOPAL DOPAC
*#
scramble siRNA
ISCHEMIA
Control
+ 0.5 mMPargyline
N. Kaludercic et al., Circ Res 2010
ischemia/reperfusionoxidative stress(toxic compounds, apoptogenic
stimuli, inflammation)
contractile dysfunction
release of catecholamines
from endogenous stores
p66Shc translocationinto mitochondria
increased MAO activity
decreasedantioxidantdefenses
PTP opening
cell death
increased mitochondrial ROS formation
oxidation of myofibrillar proteins
?
activation of signallingpathways
(e.g., PKCb)
hypertrophy
failure
Mitochondria (by means of MAO) amplify ROS formation
DOXORUBICIN administration
Increased mitochondrialROS generation and/or
decreased mitochondrial antioxidant defenses
Redox cycling by means of interaction with complex I
Mitochondrial localizationand accumulation
- doxorubicin
Hyp
erFl
uore
scen
cera
tio o
xidi
ze/b
asel
ine
Time, min
0
1
2
3
30 150 270 390 510 630 750 870 990 1110 1230 1350
0
1
2
3
30 150 270 390 510 630 750 870 990 1110 1230 1350
DoxoDoxo + Parg
+ doxorubicin
10 mMH2O2
500 mMH2O2
100 mMH2O2
Doxorubicin-treatedcells are more susceptible to H2O2-induced oxidativestress and are protected by pargyline(i.e., MAO amplifiesoxidative stress)
10 mMH2O2
500 mMH2O2
100 mMH2O2 Parg
untreated
Doxorubicin-induced increase in mitochondrial ROS formation monitored by means of mitoHyPer.Protective effect by MAO inhibition (pargyline)
- pargyline + pargyline
H2O2 addition(mM)
H2O2-induced loss of viability of HL-1 cardiomyocytesis amplified by doxorubicin
in a process blunted by MAO inhibition
0 1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
Cell
death
(%
)
Time (h)
10
10 + doxo
10 + doxo + pargy
100 + doxo + pargy
0 1 2 3 4 5 6 7 8 9 101112131415
Parg
Doxo
Dept. Biomedical SciencesUniv. of PadovaMarcella Canton Roberta Menabò Nina KaludercicAndrea CarpiSara MenazzaMarika Campesan
Paolo BernardiValeria Petronilli
Dept. BiologyUniv. of PadovaLuca Scorrano
Dept. BiochemistryUniv. of GenovaEdon Melloni
IEO MilanMarco GiorgioSimon Plyte
DETECTABLE PARAMETERS OF MITOCHONDRIAL FUNCTION
ISOLATEDMITOCHONDRIA
Oxygen consumptionATP synthesisRedox changes (NAD and FAD)DYm (quantitative)Matrix volumeIon movements
ISOLATEDCELLS
Oxygen consumptionRedox changes (NAD and FAD)DYm (semiquantitative)Matrix volume
Ion movements
ISOLATEDHEART
Oxygen consumptionATP content
IN SITUHEART
Oxygen consumptionATP content
METHODS FOR DETECTING THE OPENING OF THE MITOCHONDRIAL PERMEABILITY TRANSITION PORE (PTP)
ISOLATEDMITOCHONDRIA
SwellingCa2+ retention capacity (CRC)Permeability to solutesCsA inhibitable changes
ISOLATEDCELLS
Calcein redistributionSwelling
CsA inhibitable changes
INTACTHEART
Mitochondrial NAD depletionMitochondrial accumulation of deoxyglucoseCsA inhibitable changes
Calcein - Co2+
Calcein
Calcein loading in the absence of Co2+
Co2+ addition to calcein loaded cells
ColoadingCo2+ addition after calcein loading
• Short PTP openings are detected only by trapped
calcein and may have little impact on cell viability,
while changes of TMRM distribution require longer
PTP openings, which cause release of cytochrome c
and may result in cell death.
• Modulation of PTP open time appears to be a key
element in determining the outcome of stimuli that
converge on the PTP.
Petronilli et al., J. Biol. Chem. 276:12030-4, 2001
+
_
e-H+
O2
H O2
H+
H+ADP + Pi
ATPATP
_
_
_
_ _
_
_
_
_
_
_
_
_
_
_
_
_ _
_
_
_ _
_
+
+
+
++
+
+ +
+
+
+
+
+
+
+
+
+++
+ O2 consumptionO2 consumptionDYm collapseO2 consumptionDYm collapseATP depletion
+
O2 consumptionDYm collapseATP depletion matrix swellingOMM rupture
Protein release
↑ [Ca2+]↑ ROS↓ DYm
↓ pHADPMg2+
CsA
Cyp
DK
O I/R
Cyp
DK
O C
sA
I/R
Cyp
DK
O
No
rmo
ssia
Cyp
DK
O M
div
iI/R
5000x 10000x 5000x 10000x
5000x 10000x 5000x 10000x
ISCHEMIA
REPERFUSION
low pHhigh [Mg2+ ]low ROS
Dym fallhigh Pi
Dym recoverylow Pi
pH increase[Ca2+] increasehigh ROS[Mg2+] decrease
0
4
100
0
high
low
Dym
[Mg ] i2+
mM
5 min 5 min
ANOXIA ANOXIA
Reversible damage Irreversible damage
cell
length%
ischemia/reperfusion
accumulationof metabolic
intermediates
impairmentof Ca2+
homeostasis
ROSproduction
release ofpro-apoptotic
proteins
ATPdepletion
alterations of MYOCARDIAL
function and structure
alterations of MITOCHONDRIAL
function and structure
Relationships between [Ca2+]i increaseand increased ROS formation in mitochondria
[Ca2+] ROS
cyt. c displacement
binding tocardiolipin
NOS NO Complex IVinhibition
TCA cycle e- supply to ETC
GSH reductaseinhibition [GSH]
Complex Iinhibition
PTP opening cyt. c release
ETCinhibition
adapted from P.S. Brookes et al., Am J Physiol 287:C817-C833, 2004
PTP opening
OXIDATIVE STRESS IS UPTREAM AND DOWNSTREAM OF PTP OPENING
Amplification loops linking [Ca2+]m with ROS formation and PTP opening
respiratorychain inhibition
cyt. c release
ROS formation
Dym decrease
cardiolipinoxidation
binding toIP3R
loss of Ca2+
autoinhibition
increased [Ca2+]m
increased ERrelease of Ca2+
NADrelease
220 KDa
120 KDa100 KDa
80 KDa
60 KDa
50 KDa
40 KDa
30 KDa
PM1 2
Red Ponceau
1 2
Anti-Tm immunoblot
1: C57
2: CypD-/-
The oxidation of tropomyosin ocurring upon post-ischemicreperfusion is largely reduced in mice lacking CypD