Ischemia and Reperfusion. Basic concepts

Ischemia and Reperfusion. Basic concepts

1) Myocardial Ischemia

2) Reperfusion salvage and reperfusion injury

3) Basic molecular mechanisms

4) Integrative view

5) Translational perspective

6) Conclusion

ISCHEMIA

Blood flow insufficient to sustain aerobic metabolism

ISCHEMIC HEART DISEASE

Estenosis /occlusion of epicardial coronary arteries

Subepicardial myocardium: less tissue pressure

Subencardial layer: high tissue pressure

Normal

Arteriolar R (endo) < R(epi)

Flow (endo) = Flow (epi)

Hypoperfusion

Reactive vasodilation

R(endo) = R(epi)

F(endo) < F(epi) = 1 ml/gr/min

No flow

Flow(endo) = Flow(epi) = 0



Normal



Hypoperfusion


R(endo) = R(epi)


No flow




Normal



Hypoperfusion


R(endo) = R(epi)


No flow


ISCHEMIA

STOP OXYDATIVE PHOSPHORYLATION

Anaerobicglycolysis

CATABOLITE ACCUMULATION

ATP

STOP Na+ PUMP

[H+]i

NHE

NCX

ENERGY DEPLETION

DEPHOS-PHORYLATION [Ca2+]i

CALPAIN ACTIVATION

[Na+]i

[Na+]i

OSMOLALITY Intra/Extra Cell

ACIDOSIS Intra/Extra

Cell

Ca2+

sequestration

SR and Mito

ENERGY

INTRACELLULAR

ACIDOSIS

CONTRACTILITY

Ca

OVERLOAD

T I M E

RIGOR CONTRACTURE

CHRONOLOGY OF ISCHEMIC CHANGES

Arb

itrar

yun

its

Normoxia 20 min Isch 1” 60” 120” 180”

(anoxia pH 7.4) Reperfusion

Ischemic

rigor

Hyper-

contracture

time

cell

dea

th

no reperfusion

ischemia

100%

0%





4) Integrative view


6) Conclusion

time

cell

dea

th

no reperfusion

ischemia reperfusion

thrombolytics,

PCI

100%

0%

Myocardial infarctIon after reperfusion

following 48 min of LAD occlusion

AREA AT RISK INFARCT AREA

Reperfusion injury:

cell death preventable by interventions applied at

the time of reperfusion

time

cell

dea

th

no reperfusion

ischemia reperfusion

thrombolytics,

PCI

thrombolytics,

PCI with

cardioprotection

intervention

100%

0%





4) Integrative view


6) Conclusion

Post reperfusion necrosis: “Contraction band necrosis”

srinkage (1)

(1) Barrabés et al. Pflüger Arch 1998

Reperfusion after 48 min coronary occlusion in the anesthesized pig

CORONARY OCCLUSION

End-Diastolic

segment length

Normoxia 20 min Isch 1” 60” 120” 180”

(anoxia pH 7.4) Reperfusion

Ischemic

rigor

Hyper-

contracture

REPERFUSION

Na+ overloadNa+ / Ca2+ exchange

ATP synthesis

Ca2+ overloadSR Ca2+ uptake

SR

Ca2+

SERCA

PL

SR

Ca2+

SERCA

RyR

PLCa2+

SR

Ca2+

SERCA

RyR

PLSR

SERCA

RyR

PL

Ca2+

SERCA

RyR

PLCa2+

Ca2+

Time (min)

Fura-2 rati

o (340/380

)

0.0

0.5

1.0

1.5

2.0

2.5

Ca2+ oscillations during reoxygenation

Time (min)

Fura-2 rati

o (340/380

)

0.0

0.5

1.0

1.5

2.0

2.5Control

KB 15µM

1 2 3 4 5

1 2 3 4 5

Time (min)

340/380 fura.2 ratio (a.u)

Reoxygenation

2.0

1.0

0.034

0/3

80

Fu

ra 2

rat

io (

a.u

.)

REPERFUSION


ATP synthesis


Ca2+ oscillations/waves

SR Ca2+ release

REPERFUSION


Hypercontraction

CELL DEATH

Sarcolemmal rupture

ATP synthesis



SR Ca2+ release

40 min

PC

r (%

)

0

20

40

60

80

100

LVE

DP

(mm

Hg)

0

20

40

60

80

100

LDH

(U/m

in/g

dw)

81012141618

Energetic recovery of reperfused myocardium

Mechanism of cell death during reperfusion: contration band necrosis

40 min

PC

r (%

)

0

20

40

60

80

100

LVE

DP

(mm

Hg)

0

20

40

60

80

100

Time (s)

0 200 400 600 800 1000 1200 1400 1600 1800 2000

LDH

(U/m

in/g

dw)

02468

1012141618



40 min

PC

r (%

)

0

20

40

60

80

100

LVE

DP

(mm

Hg)

0

20

40

60

80

100

Time (s)

0 200 400 600 800 1000 1200 1400 1600 1800 2000

LDH

(U/m

in/g

dw)

02468

1012141618



40 min

BDM 20 mM 5 min

PC

r (%

)

0

20

40

60

80

100

120

LVE

DP

(mm

Hg)

0

20

40

60

80

100

120

Time (s)0 200 400 600 800 1000 1200 1400 1600 1800 2000

LDH

(U/m

in/g

dw)

02468

1012141618



40 min

BDM 20 mM 5 min

coronary artery occlusion in pigs

50 min LAD occlusion and four hours of reperfusion in the in situ pig heart.

SELECTIVE contractile blockade at the time of reperfusion

June 2011

REPERFUSION


Hypercontraction

CELL DEATH

Sarcolemmal rupture

ATP synthesis



SR Ca2+ release

Thapsig

Ryanod

PKG

SR blockers

Siegmund et al. Circulation 1997

Abdallah et al. Cardiovasc Res 2007

REPERFUSION


Fragility

Calpainactivation

Hypercontraction

CELL DEATH

Sarcolemmal rupture

ATP synthesis



SR Ca2+ release

Na+/K+-ATPase

calpain

Nx IR PC

fodrin

Nx IR PC

Na+/K+-ATPase

ankyrin

Nx IR PC

fodrin

calpain

REPERFUSION


Fragility

Calpainactivation

Hypercontraction

CELL DEATH

Sarcolemmal rupture

ATP synthesis



SR Ca2+ release

Na+ pump failure

REPERFUSION


Fragility

Calpainactivation

Hypercontraction

CELL DEATH

Sarcolemmal rupture

ATP synthesis



SR Ca2+ release

Na+ pump failure

Balut C, Kidney International 73, 226-232

Balut C, Kidney International 73, 226-232

Ca2+

ATP

ROS

pH

CsA

MPTP

Crompton

Halestrap

Griffiths

Bernerdi

DiLisa Weiss

Nature 2005; 434:655 and 658

REPERFUSION


MitochondrialPermeability

Transition

ROS

Fragility

Calpainactivation

Hypercontraction

CELL DEATH

Sarcolemmal rupture

ATP synthesis



SR Ca2+ release

Na+ pump failure

Inserte et al Circ Res 2005

Inserte et al Cardiovasc Res 2006

REPERFUSION



Transition

ROS

Fragility

Calpainactivation

Hypercontraction

CELL DEATH

Sarcolemmal rupture

ATP synthesispHinormalization



SR Ca2+ release

Na+ pump failure



Delay in pHi recovery explains postconditioning protection

Inserte et al. Cardiovasc Res 2008

control

heptanol

C1

C2

C1

C2

Garcia-Dorado et al. Circulation1997Ruiz-Meana et al. Circ Res 1999

Na+ Na+

Ca2+

REPERFUSION



Transition

ROS

Fragility

Calpainactivation

Hypercontraction

CELL DEATH

Sarcolemmal rupture




SR Ca2+ release

Na+ pump failure



Cell-to-cellpropagation

Ruiz-Meana et al. Basic Res Cardiol 2007

0 min

Ca2+ 5mM

D(TMRE)

25µmCa2+ (FLUO-4)

13 min 15 min10 min4 min

Induction of MPT in Ca2+ overloaded cells causes hypercontracture:

Induction of MPT causes HC in Ca2+ overload

REPERFUSION



Transition

ROS

Fragility

Calpainactivation

Hypercontraction

CELL DEATH

Sarcolemmal rupture




SR Ca2+ release

Na+ pump failure

Ruiz-Meana et al.

Basic Res

Cardiol 2007

Yoshikane H. J Submicrosc Cytol 1986

SR-mitochondria connection through Ca2+ microdomains

J Biol Chem 2006;281:1547

J Biol Chem 2008;283:32771

Nature 2008;456:605

Ryanodine/ Thapsigargin

Calcein 20 min SI, pH 6.4 15 R, pH 7.4

SR blockade prevents MPT in reperfused myocytes

Ryano/Tg

R-CsA

Osmotic swelling

R

10000 200 400 600 8000

20

40

60

80

100

120

Cal

cein

(a.

u.)

Time (s)

*

R R-CsA

0

10

20

30

40

50

LDH

(%

of t

otal

)

* p<0.05

Nx

** p<0.01

Ruiz-Meana et al. Am J Physiol 2009

Ryano/Tg

**

SR blockade reduces

mitochondrial damage and cell

death

Ryanodine/ Thapsigargin

0

10

20

30

40

50

LDH

rel

ease

(%

of t

otal

)

* p=0.03

*

with colchicine

R Ryano/Tg R Ryano/TgNx Nx

With colchicine

Ruiz-Meana et al. Am J Physiol 2009

Cal

cein

rel

ease

(%

of m

ax)

0

20

40

60

80

100

Ryano/Tg

with colchicine

Nx R

REPERFUSION



Transition

ROS

Fragility

Calpainactivation

Hypercontraction

CELL DEATH

Sarcolemmal rupture




SR Ca2+ release

Na+ pump failure

Ruiz-Meana et

al. Am J Physiol

2009

REPERFUSION



Transition

ROS

Fragility

Calpainactivation

Hypercontraction

CELL DEATH

Sarcolemmal rupture




SR Ca2+ release

Na+ pump failure

Inserte 2011 unbublished

MitoSR

Ca2+

SERCA

RyRC U

NCX

PL

Ca2+

Ca2+

Ca2+

oscillations MPT

REPERFUSION

Ca2+ overload ATP pH correction ROS

CELL DEATH





4) Integrative view


6) Conclusion

Wt CyD-KO0

300

Tim

e to

rig

or

on

set

(s)

p<0.01

*

Wt CyD-KO

Hyp

erco

ntr

acte

d

cells

(%

)

0

100

Td

eath

cel

ls

tryo

an b

lue

(%)

Wt CyD-KO

0

100p<0.01

*

15 min 25 min

Duration of ischemia

Effect of CyD abblation on hypercontracture and cell death during

reperfusion after short (15 min) or more prolonged ischemia in adult

cardiomyocytes

Ruiz-Meana 2010, submitted

30 60

Hyp

erc

on

trac

ture

(m

mH

g)

0

20

40

60

80

100

30 60

Ischemia (min)

LV

devP

(m

mH

g)

0

10

20

30

40

50WTCyD-KO

Ischemia (min)

LD

H (

U/g

dw

/60

min

)

0

100

200

300

400WTCyD-KO

Infa

rct

(%)

0

20

40

60

80

WTCyD-KO

WTCyD-KO

*

*

*

*

* *

Effect of CyD abblation on hypercontracture, functional recovery

and cell death during reperfusion after short (30 min) or more

prolonged (60 min) ischemia in perfused mice hearts


A)

C)

Ischemia 30min Ischemia 50min

LDH

(U

/60m

in/g

dw)

0

200

400

600

800

1000

1200

*

Control CsA BDM

Infa

rct (

%)

0

20

40

60

80

*

Hyp

erco

ntra

ctur

e (m

mH

g)

0

40

80

120

160

*

*

Control CsA BDM

*

**

*

*

B)

Effect of MPT inhibition and contractile blockade infarct size after

short (30 min) or more prolonged (50 min) ischemia in perfused rat

hearts


A)

C)

Ischemia 30min Ischemia 50min

LDH

(U

/60m

in/g

dw)

0

200

400

600

800

1000

1200

*

Control CsA BDM

Infa

rct (

%)

0

20

40

60

80

*

Hyp

erco

ntra

ctur

e (m

mH

g)

0

40

80

120

160

*

*

Control CsA BDM

*

**

*

*

B)

Effect of MPT inhibition and contractile blockade infarct size after

short (30 min) or more prolonged (50 min) ischemia in perfused rat

hearts


No-reflow/ hemorrageEdema Infarct

No-reflow

T2-STIR Late enhancement

Protecting the heart in acute ischemic syndrome Mechanism of cell death during reperfusion: non-cardiomyocyte cells





4) Integrative view


6) Conclusion

The effect of STEMI, and CAD, on survival and quality of life is mediated by cell death causing contractile failure, LV remodeling, and arrhythmias

There is one opportunity for patients with to scapean adverse evolution: EARLY reperfusion

Results of current reperfusion therapy

Very few pts arrive early enough to PCI as to abort MI

During the initial 6h of ischemia myocardial salvage is larger than in most other animal species

Even patients arriving early end-up with significant infarcts

Interventions that would enhance myocardial salvage when applied at the time of reperfusion strongly needed

Conclusion

1) The extent of ardiomyocyte death secondary to transient coronary occlusion depends mainly on duration of ischemia and residual flow (incomplete occlusion, collaterals), and progresses from endo to epicardium

2) During reperfusion, myocardial cell death occurs mainly during the first minutes after reflow, as necrosis, not apoptosis, that may propagate througgap junctions

3) Part of cell death may be prevented by interventions applied at the time of reperfusion (lethal reperfusion injury)

4) Altered Ca2+ handling leading to protease activation and hypercontracture, and mitochondrial permeability transition are main, interconnected determinants of cell death whose relative importance appears to depend on the severity of prior ischemic insult. Both depend on on pH normalization

5) No-reflow is mostly a consequence and a marker rather than a cause of large infarcts

6) Prevention of reperfusion injury in patients appears feasible an constituesthe next frontier in the treatment of patients with STEMI

David Garcia-Dorado, MD, PhD

Marisol Ruiz-Meana, DVM, PhD

Jose A. Barrabés, MD, PhD

Javier Inserte, ScD, PhD

Luis Agulló, ScD, PhD

Antonio Rodríguez-Sinovas, DVM, PhD

Ignasi Barba, ScD, PhD

Diego López, ScD, PhD

Elisabet Miro-Casas, ScD

Carmen Sartorio, ScD, PhD

Víctor Hernando, ScD

Esperanza Agulló, ScD

Angeles Rojas, Technician

Angeles García, Technician

Adoración Quiroga, Technician

Giuliana Ríos, Assistant

Mª Ángeles Carmona, ScD, PhD

Elena Abad, ScD, PhD

Celia Fernández-Sanz, ScD

Alejandra González, ScD

José Antonio Sánchez, ScD

Marcos Poncela, ScD

Eduardo Villacorta, MDLaboratory of Experimental Cardiology, Heart DepartmentVall d’Hebron University Hospital and Research InstituteUniversitat Autònoma de Barcelona

www.cardioexperimentalvh.com

Marisol Ruiz-Meana

THANK YOU

Documents

Ischemia and Reperfusion. Basic concepts