PD Dr. Kerstin Boengler

Physiologisches Institut

Oxidant stress: Consequences for

ischemia/reperfusion

ROS

ROS

Reactive oxygen species (ROS) in ischemia/reperfusion injury (IRI)

GOOD:Endogenous

protectioncontribute to

BAD:Infarction contribute to

Burns et al., J Am Coll Cardiol 39:30–36, 2002

6 Monthmortality(%)

Infarct size (% area at risk)

Myocardial infarction and prognosisPatient

Giorgio et al., Nat Rev Mol Cell Biol, 8:722-728, 2007

Potential sources of ROS

; Akki et al., J Mol Cell Cardiol 47: 15–22, 2009

Mitochondria

Other cell types

p66

SOD1

Cardiomyocytes

SOD2

CAT

Becker et al., Am J Physiol 277 Heart Circ Physiol 46: H2240–H2246, 1999

Complex I inhibitors

Formation of ROS during ischemia

Rat cardiomyocytes

NOX inhibitorNOS inhibitor

Cytosolic SOD inhibitor

ROS formation during (low-flow) ischemia

appears to be primarily caused by mitochondria!

Bolli et al., Proc Natl Acad Sci USA 86: 4695-4699, 1989

Formation of ROS during ischemia/reperfusion

Ischemia

Dog

Reperfusion

contribute to cellular protection

MorphologyFunction

contribute to cellular damage

MorphologyFunction

(Ischemia/Reperfusion Injury)(Postischemic Reperfusion,Heart Failure)

ROS during IRI

Mice

NOX2, NOX4 and irreversible IRI

Matsushima, Circ Res 2013

24 h I/R

NOX2, NOX4 and irreversible IRI

Matsushima, Circ Res 2013

systemic NOX2 KO mice cardiac NOX4 KO mice

MAO

p66Shc ROSPKCβ

MPTPp66Shc

Cytc

ROSCypD

CypD

ox ox

MAO

p66Shc ROSPKCβ

MPTPp66Shc

Cytc

ROSCypD

CypD

MAOMAO

p66Shcp66Shc ROSPKCβPKCβ

MPTPp66Shcp66Shc

Cytc

ROSCypD

CypD

ox ox

Cx43

Giorgio et al., Nat Rev Mol Cell Biol, 8:722-728, 2007

Mitochondria

p66

CardiomyocytesMice

Mitochondria-derived ROS

Normoxia

0

550

600

650

700

750

800

850

900

ControlClorgyline50 mM

Fluo

resc

ence

inte

nsity

(AU

)IR Clorgyline

25 mM

ROS formation by MAO during

ischemia/reperfusion

Ischemia/Reperfusion

Clorgyline 50 mM

Clorgyline 25 mM

Rat

Mitochondria-derived ROS

Fabio di Lisa

RV

WT

liver

WT

liver

p66S

hc-/-

RV

p66

Shc-

/-

p66

p52p46

Ponceau S

A

B

GAPDH

MnSOD

HDAC2

Serca2a

Na+/K+-ATPase

24 kDa

36 kDa

70 kDa

110 kDa

110 kDa

RV

cont

rol

H2O

2

mito

p66p52p46

RV

cont

rol

H2O

2MnSOD24 kDa

50 kDa

mitoC

RV

WT

liver

WT

liver

p66S

hc-/-

RV

p66

Shc-

/-

p66

p52p46

Ponceau S

A

RV

WT

liver

WT

liver

p66S

hc-/-

RV

p66

Shc-

/-

p66

p52p46

Ponceau S

A

B

GAPDH

MnSOD

HDAC2

Serca2a

Na+/K+-ATPase

24 kDa

36 kDa

70 kDa

110 kDa

110 kDa

RV

cont

rol

H2O

2

mito

p66p52p46

RV

cont

rol

H2O

2MnSOD24 kDa

50 kDa

mitoC

MiceStress-induced p66Shc translocation

Mitochondria

p66

*P<0.05

0

10

20

30

40

50

60

70

80

90

-/-0

10

20

30

40

50

60

70

80

90

Wildtyp p66shc -/-

Infarct size (% area at risk)

Mice

p66shc and irreversible IRI

Giorgio et al., Nat Rev Mol Cell Biol, 8:722-728, 2007

Mitochondria

SOD1

Cardiomyocytes

SOD2

CAT

Loor et al., Biochem Biophys Acta 2011

Cardiomyocytes

Mice

Catalase (CAT), superoxide dismutase (SOD) and irreversible IRI

Mitochondria

GSH

Mice

GSH during ischemia/reperfusion

Pig

Kupatt et al., Cardiovasc Res 61: 530-537, 2004

GSH and irreversible IRI

BBA 2011

Cell (Cardio)-protection

mRNA

ROS and mitochondrial morphology

Loor et al., Biochem Biophys Acta 2011

Mice

ROS and mitochondrial morphology

Kuznetsova et al, Int J Biochem & Cell Biol 41: 1928–1939, 2009

ROS and mitochondrial morphology

Mice

Mitochondrial fission and IRI

Mdivi: mitochondrial division inhibitor

Cel

ls w

ith e

long

ated

mito

chon

dria

(%)

Vector control Mfn1 Mfn2 DrpK38A hFis1

Ong et al., Circulation 2010

Li et al., PLoS Genetics 6: e1000795, 2010

ROS and mitochondrial morphology

Chen et al., Cardiovasc Res 84: 91-99, 2009

OPA1, mitochondrial morphologyand IRI

contribute to cellular protection

MorphologyFunction

contribute to cellular damage

MorphologyFunction

(Ischemia/Reperfusion Injury)(Postischemic Reperfusion,Heart Failure)

ROS during IRI and protection from it

Coronary occlusion

Control

Precon(Ischemia/Drugs)

60 min

3h5 10 min

3h

Infarct size(% area at risk)

control Precon

30

25

10

20

15

5

0

P<0.05

Ischemic preconditioning

Zhao et al., Am J Physiol 285: H579-H588, 2004

Dog

Garciarena et al., J Appl Physiol 105: 1706-1713, 2008

ROS and ischemic preconditioning

Bell et al., FASEB J 2005

Mice

NOX2-KO

NOX2, ROS and ischemic preconditioning

Heusch, Boengler, Schulz, Circulation 118: 1915-1919, 2008; Boengler, Schulz, Heusch, Cardiovasc Res 2009

PI3K

ROS

PKG

mitochondrium

p38

adenosinebradykininopioids, UCN

gp130 TNF-R

JAK

KATP

PKC

mitochondrium

Akt ERK

P70S6K

GPCR PI3K

sGC

Akt

eNOS

NO

eNOS

NO

ANPBNP

GSK3β

aldose reductase

nucleusmitochondrium

STAT3 NO

pGC

MPTP

onlyadenosine H11K

iNOS

MPTP

MnSODSIRT1

CB-R

AMPK

: age-dependent: species-dependent

NPR

Preconditioning: Signal transduction

Cx43

Boengler et al., Cardiovasc Res 67: 234-244, 2005

Boengler et al., Basic Res Cardiol 104: 141-147, 2009

Rodriguez-Sinovas et al., Circ Res 99:93-101, 2006

Heinzel et al., Circ Res 97:583-586, 2005

Görbe et al., Am J Physiol Heart Circ Physiol. 2011 Mar 11

2,5µm

Cytochrome C Connexin 43 Overlay

Mitochondria from human LV tissue

0%

10%

20%

30%

40%

50%

60%

MTR fluorescence [% above control]

WT0

10

20

30

40

50

60

70

80

0

10

20

30

40

50

60

70

80

*p<0.05

Infarct size (% area at risk)

Heinzel et al., Circ Res 97:583-586, 2005

Knockout mice

WT Cx43+/−

PLA Diaz DiazPLACx43 +/−

DiazoxideCx43 +/−

Menadione

*

Mena

Connexin 43 (Cx43), ROS and ischemic preconditioning

Mitochondria and stem cell differentiation

Lu et al., Cardiovasc Res 88: 277-286, 2010

Mitochondrial Cx43 and stem stell protection

Coronary occlusion

Control

Precon(Ischemia/Drugs)

Postcon

60 min

3h5 10 min

3h

3h

Infarct size(% area at risk)

control Precon Postcon

30

25

10

20

15

5

0

P<0.05

Ischemic pre- and post-conditioning

Zhao et al., Am J Physiol 285: H579-H588, 2004

Dog

Ischemic Postconditioning: Signal transduction

Ovize et al, Cardiovasc Res 87: 406-423, 2010

MKP-1

ROS scavenging and ischemic postconditioning

Rat

Kin et al., Shock 2008

Detection of superoxide anions

Mice

Tsutsumi et al., Life Sci 81: 1223-1227, 2007

ROS scavenging and ischemic postconditioning

contribute to cellular protection

MorphologyFunction

contribute to cellular damage

MorphologyFunction

(Ischemia/Reperfusion Injury)(Postischemic Reperfusion,Heart Failure)

ROS during IRI and protection from it

Minners et al., Cardiovasc Res 47: 68-73, 2000Yue et al., Am J Physiol 281: H590-H595, 2001

Rat heart in vitro

Mitochondrial uncoupling,ROS and IRI

P<0.05

0

5

10

15

20

25

30

35

Placebo IP10 Ascorbic acid IP10 + Ascorbic acid

Infa

rct s

ize

[% a

rea

at ri

sk]

ROS scavenging and ischemic preconditioning

Aldolase reductase and irreversible IRI