40 min

1
ischemic preconditioning
40 min
50
dogs
40
30
Infarction of the Ischemic Zone
20
10
0
Control
Murry et al, Circulation 1986741124-1136
2
IPC is receptor mediatedand involves PKC
BRADYKININ
OPIOIDS
ADENOSINE
EXTRACELLULAR
B2
A1
CYTOSOL
PKC
AMP
mPTP
ATP
3
FREE RADICALS CONTRIBUTE TO IPC
MPG N-(2-mercaptopropyonyl)-glycine
In situ rabbit hearts
INFARCTION OF RISK
Control IPC MPG IPCMPG
Baines et al J Mol Cell Cardiol. 29207, 1997
4
ROS prior to ischemia precondition the heart by
activating PKC
Isolated rabbit hearts
150
0
30
Reperfusion
Poly
INFARCTION OF RISK
Control
Hypo
XO
Hypo XO
Baines et al J Mol Cell Cardiol. 29207, 1997
5
Vanden Hoek et al. Circ Res. 86541-8. 2000
6
Where does the protective reactive oxygen species
(ROS) come from?
7
Adult Rabbit Ventricular Myocytes
We can observe ROS production
Control
ROS detected by Reduced MitoTracker, a
ROS-sensitive fluorochrome
Diazoxide
8
Bradykinin Causes ROS Generation
Adult Rabbit Cardiomyocytes
200
150
ROS Production ( of Control)
100
50
0
Control
Oldenburg et al. Cardiovasc Res. 55544-52, 2002
9
Myxothiazol blocks protection from ischemic
preconditioning in isolated hearts
Yue et al. Am J Physiol Heart Circ Physiol 281
H590-H595, 2001
10
EXTRACELLULAR
PLC
CYTOSOL

PKC
mPTP
11
Are the signaling ROS made during IPCs ischemic
phase its reperfusion phase, or the index
ischemia?
Index ischemia
IPC
12
Using the fluorochrome dihydroethidium (H2O2
sensitive) ROS appear to be made only during
ischemia
Kevin et al.
13
Isolated Rabbit hearts
Ischemia
Reperfusion
Group 1
IPC
Group 2
Group 3
150
-10
-20
-15
-5
0
5
30
Time (min)
MPG infusion
Myocardial MPG Conc.
Ischemia
Hypoxic buffer

14
Redox signaling occurs during reperfusion
100
80
Only MPG during early reperfusion blocks
protection
60
Infarction ( of risk zone)

40

20






0
IPC
Control
MPG in Reperf. phase
MPG in ischemia phase
MPG in index ischemia
Dost et al. Basic Res Cardiol 103378384 (2008)
15
The free radical mechanism explains why receptor
population during ischemia without brief
reperfusion does not protect
protection
Receptors populatedmKATP open
no ROS and no protection
Dost et al. Basic Res Cardiol 103378384 (2008)
16
Redox signaling is important in triggering
preconditioning. Could it be involved in
mediating the protection at reperfusion?
That seems unlikely because reperfusion is when a
toxic level of ROS are generated
17
ROS signaling is required for IPCs protection at
reperfusion following the index ischemia
60
50
300 uM MPG
40
Infarction ( of risk zone)
30
20
10
0
Control
IPC
IPC MPG
Liu et al. Basic Res Cardiol. 10354, 2008
18
PKC signaling is also required at reperfusion
60
50
PKC is required
40
Infarction ( of risk zone)
30
Is PKC activated at reperfusion by ROS?
20
10
0
Control
Liu et al. Basic Res Cardiol. 10354, 2008
19
Redox signaling must occur upstream of PKC
60
50
50 pM PMA
40
Infarction ( of risk zone)
30
ROS must be upstream of PKC
20
10
0
Control
Liu et al. Basic Res Cardiol. 10354, 2008
20
ROS activates PKC both prior to ischemia and
again at reperfusion
BRADYKININ
OPIOIDS
EXTRACELLULAR
B2
PLC
CYTOSOL

PKC
PLD
mPTP
OXYGEN
RADICALS
Mediators(act at reperfusion)
Triggers(act prior to ischemia)
K
ATP
CHANNEL
MITOCHONDRION
21
Anti oxidants have been ineffective in limiting
infarction in clinical trails. Many AMI patients
are unknowingly preconditioned (drugs, angina,
stuttering reperfusion, etc). Perhaps ROS
scavengers were protecting some
non-preconditioned patients but blocked IPCs
powerful protection in others.
22
FellowsTurhan DostYanping LiuXi-Ming Yang
Chris Baines Olaf Oldenburg
CollaboratorsMichael V. Cohen Stuart Critz
Stathis Iliodromites
23
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24
N-(2-mercaptopropionyl)-glycine (MPG), The
scavenger that we used to block IPCs
protection,does not scavenge H2O2 or
superoxide. It will scavenge OH and peroxynitrite
.
25
Free radicals contribute to a reperfusion injury
yet reperfusion with anti-oxidants have failed to
consistently limit infarction in lab animals or
patients. WHY?