Mitochondria in ischemia and reperfusion

Fabio Di Lisa

Department of Biomedical SciencesUniversity of Padovadilisa@bio.unipd.it

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