Download Superoxide Dismutase Plus Catalase Improve Contractile Function

148
Superoxide Dismutase Plus Catalase Improve
Contractile Function in the Canine Model of the
"Stunned Myocardium"
Karin Przyklenk and Robert A. Kloner
From the Department of Medicine, Harvard University and Brigham and Women's Hospital, Boston, Massachusetts, and Department of
Medicine, Wayne State University and Harper Hospital, Detroit, Michigan
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SUMMARY. Fifteen minutes of coronary occlusion followed by reperfusion does not result in
myocardial necrosis; however, the contractile function and high energy phosphate content of the
previously ischemic myocardium remains depressed or 'stunned' for several hours to days after
reperfusion. Oxygen-derived free radicals have been implicated in ischemia and reperfusioninduced injury in a variety of tissues. We wished to determine whether administration of free
radical scavengers superoxide dismutase plus catalase before and during occlusion, and throughout
reperfusion, could attenuate the 'stunning' produced by 15 minutes of left anterior descending
coronary artery occlusion in anesthetized, open-chest dogs. Segment shortening in the previously
ischemic zone recovered to within only ±10% of preinfusion values in the control group during
3 hours of reperfusion, while, in the treated dogs, segment shortening returned to a maximum of
56 ± 16% of preinfusion at IV2 hours post-reperfusion (P < 0.0003 compared to controls).
Similarly, superoxide dismutase + catalase-treated dogs exhibited improved wall thickening during
reperfusion (+30% to +70% of preinfusion values), compared to controls (0% to +10%). However,
this improvement in contractile function in the treated group was not accompanied by increased
adenosine triphosphate stores in the previously ischemic zone (31.8 ± 0.8 vs. 28.2 ± 2.2 nmol/
mg protein for control vs. treated groups). Infusion of superoxide dismutase + catalase did not
influence blood flow during occlusion or reperfusion. However, the treated group did exhibit a
significant decrease in blood pressure during reperfusion. Hypotension during reperfusion did
not appear to be the cause of the improved contractile function, as administration of sodium
nitroprusside (an afterload-reducing agent with no free radical scavenging properties) to an
additional group of dogs during reperfusion had no significant effect on segment shortening in
the previously ischemic tissue. Thus, treatment with free radical scavengers significantly enhanced
function, but did not improve high energy phosphate content, in the stunned myocardium. (Circ
Res 58:148-156, 1986)
BRIEF occlusion of a coronary artery (<20 minutes
in duration) followed by reperfusion does not result
in myocardial necrosis (Jennings, 1969; Deboer et
al, 1980; Kloner et al., 1983); however, the regional
contractile function and biochemical properties of
the previously ischemic tissue remain depressed for
prolonged periods of time following reperfusion
(Heyndrickx et al., 1975, 1978; Deboer et al., 1980;
Braunwald and Kloner, 1982; Kloner et al., 1983).
Whereas the myocardium eventually recovers fully
from a brief ischemic episode, the mechanism for
this transitory 'stunning* remains unresolved
(Braunwald and Kloner, 1982).
Recent indirect evidence has suggested that the
cytotoxic, oxygen-derived free radicals, Oj (the superoxide ion), 'OH (the hydroxyl radical), and
their intermediary, H2O2 (peroxide), are generated
at an accelerated rate upon reperfusion (Fig. 1).
These radicals are thought to be responsible, at least
in part, for ischemia and reperfusion-induced injury
in a variety of biological tissues, including the myo-
cardium (Parks et al., 1982; McCord, 1984; Jolly et
al., 1984; Burton et al., 1984). Specifically, administration of enzymatic free radical scavenging agents
significantly reduced the extent of necrosis produced
by 60-90 minutes of coronary artery occlusion followed by reperfusion (Chambers et al., 1983; Jolly
et al., 1984). Myocytes salvaged by free radical
scavengers upon reperfusion had been reversibly
injured by the 60-90 minutes of ischemia, and thus
may have properties similar to myocytes 'stunned*
by brief periods of occlusion followed by reperfusion. That is, the toxic action of free radicals may
offer an explanation for the prolonged depression
in function and metabolism which follows occlusions of less than 20 minutes. The objective of the
current study was to ascertain whether administration of the free radical scavengers superoxide dismutase (SOD: an enzyme catalyzing the conversion
of O? to H2O2) and catalase (accelerating the reaction of H2O2 to H2O and O2) could improve contractile function in the post-reperfused, stunned
Przyklenk and Kloner/Ettect of Free Radical Scavengers on Stunned Myocardium
I ISCHEMIAS Decrease inATP content:
degraded into
AMP
I
Adenoane
I
Inosine
Hypoxanth'ne
=- Influx of
intracelUar
Activated
protease
Xanthine
dehydrogenase
Xanthine
oxidase
Xanthine
tREFEfffuaONl—-02
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2H2O * 0 2
FIGURE 1. Chain of events leading to the production of O2-derived
free radicals (02, OH, and H2Ot) upon reperfusion of ischemic
myocardium.
myocardium following 15 minutes of coronary artery occlusion in the anesthetized, open-chest canine
preparation. The effects of SOD + catalase upon
regional myocardial blood flow, hemodynamics, and
high energy phosphate content were also investigated. As SOD + catalase produced a concomitant
reduction in arterial pressure, the question of
whether hypotension alone can influence contractile
function of the stunned myocardium was further
addressed.
Methods
SOD + Catalase Protocol
Twenty-eight mongrel dogs of either sex [27.2 ± 5.3 kg
(mean ± SD)] were anesthetized with an intravenous injection of sodium pentobarbital (30 mg/kg), intubated, and
ventilated with room air. After cannulas had been inserted
into the external jugular vein (for administration of drugs
and fluids) and left common carotid artery (for measurement of heart rate and arterial pressures), a left thoracotomy was performed through the 5th intercostal space. The
ribs were retracted, the heart was suspended in a pericardial cradle, and the left atrium was cannulated for infusion
of either SOD + catalase solution or saline placebo, and
injection of radioactive microspheres for measurement of
regional myocardial blood flow (RMBF). A Millar pressure
transducer was inserted into the left ventricle (LV) through
the left atrial appendage for measurement of LV pressure
and LV dP/dt.
A small segment of the left anterior descending coronary artery (LAD) was isolated, usually distal to the first
major diagonal branch. The LAD was then briefly occluded for approximately 5 seconds to delineate the extent
of cyanosis. Regional function in the soon-to-be ischemic
region was assessed using the transit time technique between ultrasonic crystals implanted in the myocardium,
the details of which have been presented previously
149
(Lange et al., 1984). One pair of crystals, used to measure
segment shortening (SS), was inserted through small scalpel incisions into the midmyocardium in the center of the
area to become ischemic. These crystals were positioned
perpendicular to the major axis of the heart, at a separation
of 6-12 mm. An additional pair of crystals for measurement of wall thickening (WT) was also placed into the
area to become ischemic: one crystal was inserted via a
small diagonal incision to the endocardium, while a larger,
disc-shaped crystal was sutured to the epicardium at the
point at which the ultrasonic transit time between the pair
was the shortest. SS, WT, arterial and LV pressures, and
LV dP/dt were monitored continuously during the experiment on a Gould recorder.
After the initial measurement of SS, WT, heart rate, and
arterial pressures under control conditions, dogs recieved
either SOD (5 mg/kg per hour) + catalase (5 mg/kg per
hour) dissolved in 500 ml of saline, or saline alone (500
ml). Infusion of the saline or SOD + catalase solution was
begun 15 minutes before occlusion, and was maintained
at a constant rate of approximately 2.4 ml/min for the
duration of the experiment. Measurements of hemodynamics, SS, and WT were repeated at 13 minutes postinfusion, before occlusion.
After a bolus dose of lidocaine (1.5 mg/kg, iv) had been
administered, the LAD was occluded using two Schwartz
atraumatic vascular clamps. Hemodynamics and function
were monitored at 5, 10, and 14 minutes post-occlusion,
and microspheres for measurement of RMBF were injected
at 10 minutes post-occlusion. Fifteen minutes after occlusion, the LAD was reperfused by releasing the Schwartz's
clamps.
Infusion of the control or drug solution was continued
for 3 hours following reperfusion, with measurements of
function and hemodynamics repeated at 5, 10, 15, 20, 30,
45, 60, 90, 120, 150, and 180 minutes post-reperfusion. A
second RMBF measurement was made 2 hours after reperfusion.
Immediately prior to sacrifice at 3 hours post-reperfusion, in vivo rransmural needle biopsies were taken from
both previously ischemic and normal myocardium, divided into endo- and epicardial segments, and assayed for
adenosine triphosphate (ATP) and creatine phosphate
(CP) content (Deboer et al., 1980). The LAD then was
reoccluded, and monastral blue pigment (1 ml/kg) was
injected into the coronary vascularure via the left atrium
to delineate the in vivo area at risk (AR). The heart then
was arrested with KC1 (20—40 mEq), excised, the positions
of the crystals were marked, and the heart fixed by immersion in 10% neutral buffered formalin.
After fixation, the hearts were cut into 7-10 transverse
slices, parallel to the atrioventricular groove, approximately 5 mm thick. Correct placement of the ultrasonic
crystals was confirmed, and the basal surfaces of the heart
slices and margins of the area at risk were traced onto
acetate sheets. After the right ventricular tissue had been
trimmed off, the heart slices were weighed. For measurement of RMBF, tissue samples from both the center of the
previously ischemic region and remote normal myocardium were cut and subdivided into epi-, mid-, and endocardial segments. RMBF then was quantified by the
method of Domenech et al. (1969). Extent of the area at
risk in each tissue slice was assessed by cutting out and
weighing the tracings of the LV and AR and correcting for
the weight of the tissue slice. AR weights were summed
for each heart and expressed as a percent of the LV (AR/
LV).
150
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Sodium Nitroprusside Protocol
An additional group of 15 dogs (23.4 ± 4.4 kg) were
anesthetized and instrumented as described previously. After isolation of the LAD, ultrasonic crystals for measurement of segment shortening were placed within the
midmyocardium in the center of the area to become ischemic. Wall thickening was not assessed in this group of
dogs.
After the initial measurement of SS and hemodynamic
parameters and administration of a lidocaine bolus, the
LAD was occluded for 15 minutes and then reperfused.
At 45 minutes post-reperfusion, sodium nitroprusside
(0.75-6.5 jigAg per min, iv) was infused at a dose titrated
to reduce systolic and diastolic pressures by approximately
20%, comparable to the hypotension observed in dogs
given SOD + catalase.
The infusion was maintained for 30 minutes and then
discontinued for 30 minutes; this cycle was repeated a
total of 3 times. Heart rate, arterial pressures, and SS were
monitored continuously throughout the experiment, and
were quantified pre-occlusion, 10 minutes post-occlusion,
15, 30, and 45 minutes post-reperfusion, and at 10-minute
intervals throughout infusion/discontinuation of sodium
nitroprusside.
Ten minutes after discontinuation of the final nitroprusside challenge, the in vivo area at risk was delineated
by injection of monastral blue dye, and the hearts were
arrested with KC1, excised, and immersed in formalin. AR/
LV was quantified as described previously; however,
RMBF, ATP, and CP content were not measured in this
segment of the study.
Analysis and Statistics
Values of heart rate and systolic and diastolic blood
pressures at each sample time were obtained from a mean
of five continuous cardiac cycles. Measurements of WT
and SS were timed using LV dP/dt. End-diastolic and endsystolic lengths (EDL, ESL) and wall thicknesses (EDWT,
ESWT) were defined as the onset of the rapid rise in LV
dP/dt and the peak negative dP/dt, respectively (Lange et
al., 1984). ESL, EDL, ESWT, and EDWT were measured
from three well-separated cardiac cycles for each sample
period, and were averaged. Percent SS during the sample
periods was then calculated from the formula:
Circulation Research/Vo/. 58, No. 1, January 1986
parisons was applied to the P-values. All data are presented as mean ± SEM, and values were considered to
differ significantly if P < 0.05.
Results
SOD + Catalase Protocol
Of the 28 dogs entered into the study, two were
excluded because of development of persistent arrhythmias upon cannulation of the left atrium and
infusion of saline. In addition, six dogs died of
ventricular fibrillation, five within the first 10 minutes of LAD occlusion and one immediately upon
reperfusion. Thus, 20 dogs—eight treated and 12
controls—remained.
Area at Risk
Area at risk of infarction was 23.3 ± 1.0% of the
LV for the controls, and 23.1 ± 1.6% for the SOD
+ catalase-treated group (P = NS), indicating that
infusion of oxygen-derived free radical scavengers
did not significantly influence in vivo area at risk in
this model.
Blood Flow
RMBF in the center of the area at risk was reduced
to 0.12 ± 0.03, 0.25 ± 0.08, and 0.59 ± 0.17 ml/min
per g of tissue in the subendo-, mid-, and subepicardium of the saline controls, respectively. Flow in
the treated dogs did not differ significantly during
occlusion from that of controls [0.14 ± 0.06, 0.37 ±
0.14, 0.81 ± 0.31 (Fig. 2)]. No difference in RMBF
during occlusion was observed between the treated
and control groups in normal myocardium perfused
by the circumflex bed. At 2 hours post-reperfusion,
infusion of SOD + catalase did not significantly
affect flow in either the previously ischemic area or
normal myocardium (Fig. 2). These results indicate
that treatment with SOD + catalase did not significantly alter RMBF during occlusion or after 2 hours
of reperfusion.
Segment Shortening
and, similarly, % WT was determined using:
These values were then presented in two ways: (1) normalized to the pre-occlusion measurements of %SS and
%WT (Lange et al., 1984), and (2) normalized to the most
negative value of %SS and %WT measured during occlusion (i.e., A %SS).
In the SOD + catalase protocol, RMBF, ATP, and CP
content, and AR/LV between the treated and control
groups, were compared by paired t-test. Hemodynamics
and function for the control and treated groups were
compared by repeat measures analysis. Contrasts between
the two groups then were performed for preinfusion values (for hemodynamic variables only), and at 30, 90, and
180 minutes post-reperfusion (for both hemodynamics
and function). Bonferroni's correction for multiple corn-
Infusion of either SOD + catalase or saline solution did not significantly influence segment shortening during the initial 15 minutes prior to LAD
occlusion. In 17 of the 20 animals in the study,
segment shortening was replaced by paradoxical
systolic bulging in the ischemic region during occlusion (Fig. 3). The remaining three dogs (all controls)
demonstrated a marked reduction in segment shortening during LAD occlusion. Mean %SS for the
treated gTOup was -74.5 ± 13.4%, -69.8 ± 14.1%,
and -98.5 ± 3.8% of the preinfusion value at 5, 10,
and 14 minutes post-occlusion, while the control
dogs exhibited a mean %SS of -45.3 ± 15.0%,
-45.2 ± 17.5%, and -54.7 ± 15.0% of the preinfusion value at comparable times during occlusion
(Fig. 3). These data suggest that the severity of
ischemia, assessed by regional function, was as bad
151
Przyklenk and Kloner/Effect of Free Radical Scavengers on Stunned Myocardium
0 Treated
D Control
Area at Risk
10'Post Occlusion
Nonjschemic Tissue
2 hr Post Rtperfusion
10'Post Occlusion
2 hr Post Reperfusion
2.4
2.2
2JO
LL
m
cc
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
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0
ENDO
MID
EPI
10 Post Occlusion
FIGURE 2. Regional myocardial blood flow
(RMBF: ml/min per g tissue) in the center
of the area at risk, and in remote, nonischemic tissue during LAD occlusion and
after 2 hours of reperfusion.
i
MID
ENDO
2 hr Post Reperfusion
EPI
ENDO
10 Post Occlusion
if not worse in the treated group as compared to the
controls.
During the 3 hours of reperfusion, the 12 control
dogs demonstrated a mean %SS ranging within
approximately ±10% of the preinfusion value (1.9
± 16.0%, -3.9 ± 16.1%, and -10.1 ± 14.1% at 30
minutes, Vh hours, and 3 hours, respectively). In
spite of the severe systolic bulging demonstrated by
the treated dogs during occlusion, %SS in the SOD
+ catalase-treated group returned to 56 ± 16% of
the preinfusion value at Vh hours post-reperfusion,
and leveled off to 19.8 ± 14.6% at 3 hours postreperfusion (Fig. 3). At both Vh and 3 hours postreperfusion, %SS for the SOD + catalase-treated
dogs was significantly greater (P < 0.0003 and P <
0.047, respectively) than that of controls.
ENDO
MID
2 hr Post Reperfusion
When segment shortening data was normalized
to the most negative value during occlusion for each
dog (i.e., to compensate for variations in the degree
of passive bulging during occlusion among the animals), the saline control group showed a A %SS of
+39.7 ± 11.7% at 30 minutes, +46.5 ± 11.1% at Vh
hours, and +40.3 ± 9.1% at 3 hours post-reperfusion. Dogs treated with SOD + catalase had a A
%SS of +103.2 ± 16.0%, +140.5 ± 21.8%, and
+104.2 ± 16.7%, respectively. Differences between
the two groups were significant (P < 0.0003) at all
three time periods (Fig. 4).
+160%
+ 140%
Treoted
Control
+ 120%
£
+120%
+ 100%
+ 80%
g1
+ 60%
£
+ 40%
I
+ 100%
*P<0.047
-
2 0
80%
c
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Z
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P<0.0003
T
*
1
+ 60%
1 1
+ "0%
-40%
-60%
- 80%
-100%
0%
Pre \
I
Occlusion)
Reperfusion
2hr
- Post Reperfusion -
3hr
FIGURE 3. Segment shortening, expressed as a percent of preinfusion
values, for SOD + catalase-treated vs. control groups.
I I
I
0c,:dmion4
1
Reperfusion
1
1
1
,1 hr.
1—i
1
1
1
1
2 hr.
-Post R«p«rfirtJon-
1
1
3hr.,
3
h
FIGURE 4. Improvement in percent segment shortening, normalized
to the most negative values during occlusion, for SOD + catalasetreated vs. control groups.
Circulation Research/Vol. 58, No. 1, January 1986
152
Treoied
Control
Wall Thickening
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Fifteen minutes of SOD + catalase or saline infusion did not alter pre-occlusion wall thickening in
the soon-to-be ischemic tissue. Upon occlusion, both
treated and control dogs demonstrated passive thinning, rather than active thickening, during systole
(Fig. 5). As was the case with %SS, animals infused
with SOD + catalase demonstrated greater values
of %WT during reperfusion (+42.8 ± 10.7%, +71.0
± 15.3%, and +39.6 ± 14.0% at lh, Vh, and 3 hours)
than did the controls (+0.2 ± 12.1%, +6.0 ± 12.8%,
and +2.4 ± 13.1%, respectively). Differences between the two groups proved significant at 30 minutes and Vh hours post-reperfusion (P < 0.0027 and
P < 0.0003), but not at the end of the reperfusion
phase [P < 0.0546 (Fig. 5)].
When the baseline for wall thickening was taken
as the most negative value during occlusion, SOD
+ catalase-treated dogs exhibited a A %WT of +78.2
± 19.1%, +102.9 ± 20.1%, and +81.5 ± 20.7% at
Vi, Vh, and 3 hours post-reperfusion. In contrast,
the saline controls improved by only +27.0 ± 5.7%,
+33.0 ± 9.1%, and +29.5 ± 11.2% (Fig. 6). Differences in A %WT between the treated and control
dogs were significant at 30 minutes, Vh, and 3 hours
post-reperfusion (P < 0.0069, P < 0.0003, and P <
0.0408).
Hemodynamics
Heart rate measured preinfusion did not differ
significantly between the two groups of dogs, and
was not influenced significantly, during the course
of the experiment, by infusion of SOD + catalase
(Fig. 7). Both systolic and diastolic blood pressures
were slightly lower prior to infusion in the treated
group. Systolic and diastolic pressures were reduced
significantly during reperfusion in dogs treated with
SOD + catalase [P < 0.0004 at 30 minutes, Vh
hours, and 3 hours post-reperfusion (Fig. 7). However, there was no change in arterial pressures in
Treated
Control
1-1— -1— -1---1-—1
Pre •
I
Occlusion I
Reperfusion
lhr.
2hr.
-Post Reperfusion-
160%
140%
*P<0-0OO3
120%
*P<0.0406
*P<0.OO69i
ioo%
80%
40%
20%
0%
L
rOcclusion |
Reperfusion
2hr.
-Post Reperfusion-
3hr.,
FIGURE 6. Improvement in percent wall thickening, normalized to
the most negative value during occlusion.
the control group during the course of the experiment.
High Energy Phosphates
ATP content in normal myocardium averaged 35
nmol/mg protein in both control and treated groups.
In the area previously ischemic, ATP concentration
remained somewhat depressed, particularly in the
endocardium (i.e., approximately 80% of control)
after 3 hours of reperfusion. However, there was no
difference in ATP content between control dogs and
those infused with SOD + catalase (Fig. 8).
Mean CP concentration was 52 nmol/mg protein
in normal myocardium. Following 3 hours of reperfusion, CP content in the previously ischemic tissue
had returned to (or slightly above) normal, and was
not influenced by treatment with SOD + catalase
(Fig. 9).
Sodium Nitroprusside Protocol
Of the 15 dogs entered into this part of the study,
two were excluded because no area at risk or evidence of post-ischemic stunning could be detected.
Data from one other animal could not be validly
analyzed due to electrical interference in the segment shortening signal, and an additional six animals succumbed to ventricular fibrillation (five
during occlusion and one immediately upon reperfusion). Results for the remaining six dogs are presented below.
3hr.
FIGURE 5. Wall thickening, expressed as a percent of preinfusion
values.
Area at Risk
AR/LV averaged 20.1 ± 1.5% in the six dogs
treated with sodium nitroprusside, a value which
Przyklenk and K/oner/Effect of Free Radical Scavengers on Stunned Myocardium
153
Treated
Control
Treated
I
1 Control
Heart Rate (BPM)
170
160
150
140
130
120
Nonischtmic Truu*
70
-I
60
50
40
30
20
Systolic BP (mm Hq)
160
150
140
130
120
110
100
10
0
#P<
00024
0.0004
*P<00OO4
FIGURE 9. CP content (nmol/mg protein) in the center of the area at
risk and in distant, nonischemic tissue at 3 hours post-reperfusion.
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dogs remained similar to those in the SOD + catalase
and saline control groups (Fig. 7).
Diastolic BP (mm Hg)
Segment Shortening
Pre I
Infusion'
Occlusion
All six dogs demonstrated paradoxical systolic
bulging in the ischemic zone during occlusion. When
segment shortening for each dog was normalized to
the value measured during occlusion, A %SS averaged +55.5 ± 8.5%, and +58.4 ± 7.3%, and +56.5
Post Repertusion-
170
Reperfusion
Heart Rate (BPM)
Infusion
160
FIGURE 7. Heart rate, systolic, and diastolic blood pressures for SOD
+ catalase-treated and control groups.
nfusion
Off
I
Infusion
I Off
I
I Off
150
140
130
does not differ significantly from those of the SOD
+ catalase or saline control groups.
Hemodynamics
Both systolic and diastolic pressures were reduced
by approximately 20% during infusion of nitroprusside (Fig. 10); thus, the magnitude of the induced
hypotension was comparable to that observed in
dogs treated with SOD + catalase (Fig. 7). At these
doses, nitroprusside infusion was not accompanied
by reflex tachycardia (Fig. 10)—heart rates in these
120
160
150
140
130
120
110
100
Systolic BP (mm Hg)
WV/\ Treated
I
| Control
NomKhwnic Tltiue
130
Diastolic BP (mm Hq)
120
110
50
100
40
90
30
80
20
70
10
0
Pre + |
EN DO
Occlusion
EPI
FIGURE 8. ATP concentration (nmol/mg protein) in the center of the
area at risk and in distant, nonischemic tissue at 3 hours postreperfusion.
I
lhr.
2hr.
3hr.
-Post Reperfusion-
Reperfusion
FIGURE 10. Heart rate, systolic and diastolic blood pressures for
animals treated with sodium nitroprusside.
Circulation Research/Vol. 58, No. 1, January 1986
154
±9.4% during the three periods of nitroprusside
infusion, and +48.1 ± 9.4%, +44.4 ± 10.0%, and
+47.2 ± 10.8% while the infusion was discontinued
(Fig. 11). Values of A %SS both with and without
nitroprusside infusion do not differ from those of
the saline control group illustrated in Figure 4, and
are clearly less than the maximum A %SS of +140%
observed in animals treated with SOD + catalase.
Thus, although values of A %SS fluctuated very
slightly upon infusion and discontinuation of the
afterload-reducing agent, infusion of sodium nitroprusside did not significantly improve the contractile
function of the stunned myocardium.
Discussion
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Oxygen-derived free radicals (O2, ' OH) and H2O2
are unstable, highly reactive, and cytotoxic variations of the oxygen molecule. These reactive oxygen
species have been implicated in ischemia and reperfusion-mediated injury of a variety of tissues, including the brain, intestine, lung, kidney, and heart
(Parks et al., 1982; McCord, 1984; Jolly et al., 1984;
Burton et al., 1984). Free radicals are produced in
vivo by both mitochondria and phagocytes; however, their rate of production is greatly accelerated
upon reoxygenation of ischemic tissue (McCord et
al., 1984).
Free radical scavengers such as superoxide dismutase and catalase are the enzymatic defenses
against these cytotoxic agents. SOD acts by catalysing the dismutation of O2 to H2O2 and O2, while
catalase accelerates the conversion of H2O2 to H2O
and O2 (Fig. 1; Fridovich, 1978; Jolly et al., 1984).
However, during reperfusion of ischemic tissue, not
only is the rate of free radical production increased,
but the damage is exacerbated by the washout of
enzymes, including endogenous supplies of SOD
in
•£ «IOO%
9
i
i
O
1
L/-|
1-
Occlusion
Rcpcrfuslon
2hr.
-Po»t Repwfutioo-
FIGURE 11. Improvement in percent segment shortening, normalized
to the value measured during occlusion, for animals treated with
sodium nitroprusside.
and catalase, from the previously ischemic tissue
(McCord, 1984). Thus, reoxygenated tissue becomes
even more susceptible to free radical mediated injury.
A large body of evidence has been presented to
indicate that myocardial necrosis resulting from ischemia followed by reperfusion may be attributed
to the formation of oxygen-derived free radicals.
Using a canine model of myocardial infarction,
Chambers et al. (1983) found that administration of
either SOD or allopurinol (a xanthine oxidase inhibitor) during 1 hour of LAD occlusion and 4 hours of
reperfusion significantly reduced infarct size, compared to nontreated controls. Similar results were
obtained by Jolly et al. (1984): SOD + catalase
significantly reduced the extent of necrosis produced
by 90 minutes of circumflex occlusion followed by
22V2 hours of reperfusion. Similar results have been
obtained with isolated, perfused hearts subjected to
global ischemia followed by reperfusion: administration of free radical scavengers SOD, catalase,
allopurinol, and/or mannitol improved subcellular
and mechanical function, and qualitative morphology of the hearts (Guarnieri et al., 1980; Schlafer et
al., 1982a, 1982b; Stewart et al., 1983; Casale et al.,
1983; Gauduell and Duvelleroy, 1984). It should be
noted that in both the in vivo and in vitro models,
the protection afforded by the scavengers was significant only when administered prior to and at the
onset of reperfusion, and was not significant when
given during reperfusion alone (Casale et al., 1983;
Gardner et al., 1983; Guaduell and Duvelleroy,
1984; Jolly et al., 1984). Direct proof of the toxic
effects of the O2 and OH radicals on developed
tension and myocardial ultrastructure has been demonstrated in isolated, perfused rabbit septa (Burton
et al., 1984). These studies indicate that O2 and
OH radicals are capable of producing functional
and structural alterations in the myocardium, and
treatment with free radical scavengers can, at least
in part, ameliorate this damage.
During coronary artery occlusion, active contraction of the myocardium is almost immediately replaced by passive systolic bulging and wall thinning
in the area of ischemia (Heyndrickx et al., 1975;
Heyndrickx et al., 1978). For occlusions lasting less
than 20 minutes, followed by reperfusion, ischemic
damage to the myocytes is reversible and the cells
do not become necrotic (Jennings, 1969; Lange et
al., 1984). However, the structural, functional, and
metabolic properties of the previously ischemic tissue have been shown to remain depressed—or
'stunned*—for prolonged periods of time following
reperfusion (Heyndrickx et al., 1975; Weiner et al.,
1976; Heyndrickx et al., 1978; Deboer et al., 1980;
Braunwald and Kloner, 1982; Lange et al., 1984;
Kloner et al., 1984). In the present study, measurements of function, biochemistry and RMBF for the
control group during the 3 hours of reperfusion
indicate that the previously ischemic myocardium
was 'stunned,* and are in good agreement with
Przyklenk and Kloner/Effect of Free Radical Scavengers on Stunned Myocardium
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previous findings: %SS and %WT remained markedly depressed throughout reperfusion (as was observed by Lange et al., 1984), ATP content was
approximately 20% below normal at 2 hours postreperfusion, while CP concentration was restored to
control values following 2 hours of reflow (as shown
by Deboer et al., 1980, Lange et al., 1984b, and
Heyndrickx et al., 1978, respectively).
The phenomenon of the stunned myocardium has
been well-documented; yet, the explanation for its
occurrence remains pending. As the recovery of
function has been observed to parallel the restoration of the ATP pool, it has been suggested that
these two factors may be causally related (Braunwald and Kloner, 1982). Increased concentrations of
intracellular calcium have also been implicated as a
possible explanation for 'stunning' (Braunwald and
Kloner, 1982). Our current results indicate that treatment with SOD + catalase significantly improves
function in the previously ischemic myocardium,
implying that OJ and ' OH ions contribute to stunning during reperfusion. As illustrated in Figure 1,
free radical formation upon reperfusion is closely
related to the degradation of ATP and influx of Ca++
during hypoxia. In fact, it has been postulated that
oxygen-derived free radicals contribute to ischemiainduced damage of sarcoplasmic reticulum, and
thereby directly affect Ca transport (Hess et al.,
1981). Thus, it seems possible that all three factors
interact and contribute to depressed function during
reperfusion.
It is interesting to note that improvement in SS
and WT in the SOD + catalase-treated group was
effected without a significant change in stores of
ATP in the previously ischemic region. This dissociation in recovery of function vs. failure of recovery
of ATP concentration suggests that the stunned
myocardium is not solely a consequence of a reduced
supply of high energy phosphates. However, it
should be stressed that normal function was not
fully restored in the SOD + catalase-treated group
(%SS and %WT returned to a maximum of +60%
and +70% of control, respectively), implying that
other as yet undefined factors also contribute to the
phenomenon of stunning. Improved function also
was not accompanied by a decrease in ATP concentration in the treated dogs—that is, the enhanced
SS and WT did not appear to drain further the high
energy phosphate pool.
Both SOD and catalase are highly specific in their
roles of Oj and 'OH scavengers (McCord et al.,
1984); thus, reduction in infarct size and/or improvement in function upon treatment with these
agents has been attributed directly to their enzymatic
action. However, in the present study, animals infused with SOD + catalase demonstrated significant
reductions in arterial blood pressure during reperfusion. No such response was observed in the saline
control group. It is possible that the improved function observed during reperfusion in the present
study may be due in part to the concomitant reduc-
155
tion in afterload, rather than the elimination of free
radicals alone. To address this question, the timing
and magnitude of the hemodynamic changes observed with SOD + catalase were mimicked by
infusion of sodium nitroprusside, an afterload-reducing agent with no free radical scavenging ability.
Hypotension produced by sodium nitroprusside
during reperfusion was clearly not accompanied by
a similar improvement in contractile function as
occurred in the SOD + catalase group. These findings support our conclusion that the salutory effects
of SOD + catalase on contractile function of the
stunned myocardium are due to their free radical
scavenging action, and not a consequence of afterload reduction.
Supported in part by N1H SCOR HL-26215, Bethesda, Maryland.
Dr. Przyklenk holds a postdoctoral fellowship from the Medical
Research Council of Canada.
Dr. Kloner is an established investigator of the American Heart
Association.
Address for reprints: Dr. Karin Przyklenk, Experimental Myocardial Ischemia Lab, Harper Hospital, 3990 John R, Detroit, Michigan
48201.
Received April 29, 2985; accepted for publication October 30,
1985.
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INDEX TERMS: Myocardial ischemia • Oxygen-derived free
radical scavengers • Regional myocardial function • Sonomicrometry • Coronary reperfusion • Sodium nitroprusside
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Superoxide dismutase plus catalase improve contractile function in the canine model of the
"stunned myocardium".
K Przyklenk and R A Kloner
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Circ Res. 1986;58:148-156
doi: 10.1161/01.RES.58.1.148
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