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
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
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
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%
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
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%
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
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
Na+ overloadNa+ / Ca2+ exchange
ATP synthesis
Ca2+ overloadSR Ca2+ uptake
Ca2+ oscillations/waves
SR Ca2+ release
REPERFUSION
Na+ overloadNa+ / Ca2+ exchange
Hypercontraction
CELL DEATH
Sarcolemmal rupture
ATP synthesis
Ca2+ overloadSR Ca2+ uptake
Ca2+ oscillations/waves
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
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
Energetic recovery of reperfused myocardium
Mechanism of cell death during reperfusion: contration band necrosis
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
Energetic recovery of reperfused myocardium
Mechanism of cell death during reperfusion: contration band necrosis
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
Na+ overloadNa+ / Ca2+ exchange
Hypercontraction
CELL DEATH
Sarcolemmal rupture
ATP synthesis
Ca2+ overloadSR Ca2+ uptake
Ca2+ oscillations/waves
SR Ca2+ release
Thapsig
Ryanod
PKG
SR blockers
Siegmund et al. Circulation 1997
Abdallah et al. Cardiovasc Res 2007
REPERFUSION
Na+ overloadNa+ / Ca2+ exchange
Fragility
Calpainactivation
Hypercontraction
CELL DEATH
Sarcolemmal rupture
ATP synthesis
Ca2+ overloadSR Ca2+ uptake
Ca2+ oscillations/waves
SR Ca2+ release
Na+/K+-ATPase
calpain
Nx IR PC
fodrin
Nx IR PC
Na+/K+-ATPase
ankyrin
Nx IR PC
fodrin
calpain
REPERFUSION
Na+ overloadNa+ / Ca2+ exchange
Fragility
Calpainactivation
Hypercontraction
CELL DEATH
Sarcolemmal rupture
ATP synthesis
Ca2+ overloadSR Ca2+ uptake
Ca2+ oscillations/waves
SR Ca2+ release
Na+ pump failure
REPERFUSION
Na+ overloadNa+ / Ca2+ exchange
Fragility
Calpainactivation
Hypercontraction
CELL DEATH
Sarcolemmal rupture
ATP synthesis
Ca2+ overloadSR Ca2+ uptake
Ca2+ oscillations/waves
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
Na+ overloadNa+ / Ca2+ exchange
MitochondrialPermeability
Transition
ROS
Fragility
Calpainactivation
Hypercontraction
CELL DEATH
Sarcolemmal rupture
ATP synthesis
Ca2+ overloadSR Ca2+ uptake
Ca2+ oscillations/waves
SR Ca2+ release
Na+ pump failure
Inserte et al Circ Res 2005
Inserte et al Cardiovasc Res 2006
REPERFUSION
Na+ overloadNa+ / Ca2+ exchange
MitochondrialPermeability
Transition
ROS
Fragility
Calpainactivation
Hypercontraction
CELL DEATH
Sarcolemmal rupture
ATP synthesispHinormalization
Ca2+ overloadSR Ca2+ uptake
Ca2+ oscillations/waves
SR Ca2+ release
Na+ pump failure
Inserte et al Circ Res 2005
Inserte et al Cardiovasc Res 2006
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
Na+ overloadNa+ / Ca2+ exchange
MitochondrialPermeability
Transition
ROS
Fragility
Calpainactivation
Hypercontraction
CELL DEATH
Sarcolemmal rupture
ATP synthesispHinormalization
Ca2+ overloadSR Ca2+ uptake
Ca2+ oscillations/waves
SR Ca2+ release
Na+ pump failure
Inserte et al Circ Res 2005
Inserte et al Cardiovasc Res 2006
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
Na+ overloadNa+ / Ca2+ exchange
MitochondrialPermeability
Transition
ROS
Fragility
Calpainactivation
Hypercontraction
CELL DEATH
Sarcolemmal rupture
ATP synthesispHinormalization
Ca2+ overloadSR Ca2+ uptake
Ca2+ oscillations/waves
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
Na+ overloadNa+ / Ca2+ exchange
MitochondrialPermeability
Transition
ROS
Fragility
Calpainactivation
Hypercontraction
CELL DEATH
Sarcolemmal rupture
ATP synthesispHinormalization
Ca2+ overloadSR Ca2+ uptake
Ca2+ oscillations/waves
SR Ca2+ release
Na+ pump failure
Ruiz-Meana et
al. Am J Physiol
2009
REPERFUSION
Na+ overloadNa+ / Ca2+ exchange
MitochondrialPermeability
Transition
ROS
Fragility
Calpainactivation
Hypercontraction
CELL DEATH
Sarcolemmal rupture
ATP synthesispHinormalization
Ca2+ overloadSR Ca2+ uptake
Ca2+ oscillations/waves
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
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
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
Ruiz-Meana 2010, submitted
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
Ruiz-Meana 2010, submitted
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
Ruiz-Meana 2010, submitted
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
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
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