© 1996 American Heart Association, Inc.
Articles |
Effect of Ischemic Preconditioning of the Myocardium on cAMP
From the Division of Cardiovascular Research, Research Institute, The Hospital For Sick Children, Toronto, Ontario, Canada; the Division of Cardiovascular Surgery, Research Institute, The Toronto Hospital; and the Departments of Physiology, Molecular and Cellular Pathology and Surgery, The University of Toronto.
Correspondence to Reena Sandhu, Cardiovascular Laboratories, Room CCRW1-885, The Toronto Hospital, General Division, 200 Elizabeth St, Toronto, Ontario, M5G 2C4, Canada. E-mail r.sandhu@utoronto.ca.
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Abstract |
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 Top Abstract IntroductionMaterials and Methods Results Discussion References |
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Abstract Reduction of cAMP has been implicated in the protection of ischemic preconditioning (IP), but until now, this possibility has not been directly addressed. In this study, we found that in the in vivo rabbit heart, 10 to 30 minutes of sustained regional ischemia was accompanied by a nearly twofold rise in cAMP levels. This increase in cAMP was attenuated when sustained ischemia was preceded by IP induced with a single cycle of transient ischemia and reperfusion (TI/R) and prevented when ischemia was preceded by three cycles of TI/R. The mechanism of cAMP reduction by IP does not involve activation of protein kinase C (PKC), since the PKC inhibitor polymyxin B (24 mg/kg) did not raise cAMP levels during sustained ischemia in IP hearts. Furthermore, this effect is also not mediated by reduced responsiveness of the ß-adrenergic effector pathway, since both nonischemic hearts and hearts subjected to three cycles of TI/R exhibited similar increases in cAMP in response to 5 µg/kg isoproterenol. However, propranolol (0.75 mg/kg) abolished the rise in cAMP levels observed during sustained ischemia in control hearts but did not reduce cAMP levels further in IP hearts. These data indicate that the ischemia-induced rise in cAMP levels in control hearts was mediated by activation of the ß-adrenergic receptor. Taken together with data demonstrating that ß-adrenergic responsiveness was not affected by IP, these data support the conclusion that the lack of elevation in cAMP levels observed during sustained ischemia in IP hearts is mediated by an attenuation of norepinephrine release. To examine whether the protection of IP against necrosis was mediated by the lack of elevation in cAMP levels, we determined whether the infarct size–limiting effect of IP could be blocked by NKH477, an activator of adenylyl cyclase. Four groups of rabbits were subjected to 30 minutes of in vivo regional ischemia and 90 minutes of reperfusion. Control hearts (n=10) had 53.6±5.5% infarction of the area at risk. IP with three cycles of transient ischemia limited infarct size to 3.2±1.3% (n=13, P<.0001). NKH477 (45 µg/kg) increased average cAMP levels in IP hearts during sustained ischemia to levels similar to those in untreated control hearts. However, NKH477 did not block IP (50.2±7.7% of the area at risk was infarcted in the control+NKH477 group [n=10] versus 10.0±5.9% in the IP+NKH477 group [n=7], P<.05). Therefore, we conclude that although IP lowers cAMP levels during sustained ischemia, this effect is not necessary for its protection against necrosis, since raising cAMP does not block this protection of IP.
Key Words: preconditioning • ischemia • cAMP • adenylyl cyclase • myocardial infarction
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Ischemic preconditioning (IP) refers to the phenomenon whereby one or more periods of transient myocardial ischemia protect the heart against injury produced by a subsequent, more prolonged period of myocardial ischemia and reperfusion. The signal transduction pathways involved in producing this phenomenon are currently under investigation; but as yet, their importance remains incompletely understood. One signal transduction pathway that has been implicated in IP1 2 but has received little experimental attention is the cAMP pathway. In the present study, we show that IP induced with three cycles of transient ischemia and reperfusion prevents the nearly twofold rise in cAMP that occurs with sustained ischemia.
One goal of the present study was to investigate the mechanism by which IP prevents the ischemia-induced elevation in cAMP levels. Three possibilities were studied. The first was that activation of PKC reduces cAMP levels in IP. Evidence suggesting that PKC activation plays an important role in the protection of IP has recently emerged.3 4 There is considerable "cross talk" between the cAMP and PKC signal transduction pathways, with PKC exerting multiple effects on both the cAMP-generating and -degrading pathways in the heart, some of which are consistent with a reduction of cAMP levels.5 To investigate the importance of PKC activation in the cAMP-lowering effect of IP, we determined whether this effect could be blocked with the specific PKC inhibitor polymyxin B.
The second possibility was that the episodes of transient ischemia in IP hearts result in the liberation of norepinephrine, as well as other mediators, which then act to desensitize the ß-adrenergic effector pathway and prevent the rise in cAMP during the subsequent sustained ischemia. We focused on the ß-adrenergic effector pathway, because the ß-adrenergic receptor can desensitize fairly rapidly,6 a requirement for a role in IP. Rapid desensitization of the ß-adrenergic receptor occurs through uncoupling of this receptor from Gs proteins by multiple mechanisms, including phosphorylation of this receptor by the ß-adrenergic receptor kinase and the subsequent binding of ß-arrestin,7 activation of cAMP-dependent kinases,6 and activation of PKC.5 We reasoned that if the ß-adrenergic signal transduction pathway was desensitized by IP, then myocardium that had been ischemically preconditioned would exhibit an attenuated rise in cAMP in response to ß-adrenergic receptor stimulation with isoproterenol compared with cAMP levels in nonpreconditioned myocardium.
The third possibility was that events triggered by transient ischemia and reperfusion in IP hearts attenuate norepinephrine release during sustained ischemia and that this, in turn, leads to diminished ß-receptor activation and, consequently, reduced cAMP formation. Schömig8 has shown that in nonpreconditioned rat hearts, periods of myocardial ischemia of >10-minute duration can increase local norepinephrine concentrations within the ischemic myocardium to 100 to 1000 times normal plasma concentrations. Furthermore, this group recently demonstrated that norepinephrine release during sustained ischemia is greatly reduced by IP.9 We reasoned that if reduced stimulation of the ß-adrenergic receptor by norepinephrine was responsible for preventing the rise in cAMP levels in IP hearts, then ß-blockade with propranolol should reduce cAMP levels in control hearts subjected to sustained ischemia to levels similar to those observed in IP hearts.
Another goal of the present study was to determine whether the lack of elevation in cAMP levels in IP hearts was necessary for its protection against necrosis. Several lines of evidence are consistent with this hypothesis. First, even in the absence of ischemia, elevations in cAMP have been shown to produce myocardial necrosis.10 11 High levels of cAMP are also known to increase sarcolemmal calcium entry12 and to increase the activation of cardiac lipases,13 effects that are known to aggravate ischemic injury. Furthermore, Thornton and colleagues have demonstrated that the protective effect of IP against necrosis can be mimicked by the administration of either adenosine A1 receptor agonists14 or muscarinic M2 receptor agonists.15 In cardiac myocytes, both these receptors are coupled to adenylyl cyclase through Gi proteins,16 and stimulation of these receptors inhibits cAMP formation.17 Finally, the protection of IP against necrosis can be blocked by inactivation of Gi,15 an effect that would also be expected to increase cAMP. To test the hypothesis that IP is mediated by a reduction in cAMP levels, we treated hearts with NKH477, an activator of adenylyl cyclase, to determine whether this could block the protection of IP against necrosis in our in vivo model of regional ischemia and reperfusion in the rabbit heart.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Surgery
We used New Zealand White rabbits of either sex for the present study (weight range, 3 to 4 kg). Rabbits were treated in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
To induce anesthesia, we cannulated the marginal ear vein of the right ear for administration of sodium pentobarbital (28 mg/kg) and heparin (285 U/kg). During the experiment, anesthesia was maintained with sodium pentobarbital (25 mg/kg per hour). Anticoagulation was maintained with intravenous heparin (100 U/kg per hour). We administered both drugs diluted in 5% dextrose (25 mL/h). After the induction of surgical anesthesia, we performed a tracheotomy. We ventilated the rabbits with 100% oxygen by using a respirator (Harvard Apparatus). Respiratory rate was set at 35 breaths per minute, and tidal volume was 18 to 24 mL. We made arterial blood gas determinations periodically throughout the experiment and adjusted the tidal volume to keep PCO2, pH, and PO2 within the physiological range. We used a catheter inserted in the left carotid artery to monitor blood pressure and heart rate. For infusion of drugs, we cannulated the marginal ear vein of the left ear.
To expose the heart, we performed a left thoracotomy through the fourth intercostal space and opened the pericardium. We then passed a 2-0 polypropylene suture around the left main coronary artery and passed the ends of the suture through a flanged piece of polyethylene tubing. In experiments involving the creation of ischemia, we accomplished it by pushing the flange against the coronary artery to produce a snare and clamping it in position with a mosquito clamp. We confirmed the presence of ischemia by observing cyanosis distal to the occlusion site. Reperfusion was achieved by releasing the snare. If fibrillation occurred during the course of the experiment, a defibrillator (Burdick Co) was used.
cAMP Determination Study
cAMP Levels in Control and IP
Hearts
After the completion of surgery, animals were stabilized for 30
minutes. Rabbits were then assigned randomly into the control and IP
groups. Rabbit hearts in the IP group subsequently received three
cycles of 5-minute regional ischemia and 10 minutes of
reperfusion. Hearts in animals in the control group had an additional
45-minute stabilization period. Control and three-cycle IP hearts
were harvested for cAMP determination at the following time points:
immediately before the sustained ischemia (ie, after the
75-minute stabilization period in the control group and after three
cycles of transient ischemia/reperfusion in the IP group) and
after 10, 20, and 30 minutes of sustained ischemia (n
6 per
time point per group). An additional group of hearts was harvested
after only the initial 30-minute stabilization period (n=5) to serve as
the true baseline value for all groups. As expected, there was no
difference in cAMP levels in these hearts and those harvested after the
75-minute stabilization period; consequently, the cAMP data from these
two groups of hearts were pooled to generate a larger true-baseline
group (n=16).
To examine the effect of transient ischemia on cAMP levels, two additional groups of hearts were harvested at the end of the first and third episodes of 5-minute transient ischemia in hearts assigned to the IP protocol (n=6 per group). The group harvested after the first episode of 5-minute transient ischemia also provided the cAMP levels for 5 minutes of sustained ischemia in the control group, since both groups were subjected to a stabilization period followed by 5-minute ischemia. To examine whether the number of cycles used to induce IP affects the cAMP response during the subsequent sustained ischemia, another group of hearts (n=10) was harvested after being subjected to a single cycle of IP (5-minute regional ischemia and 10-minute reperfusion) followed by 10 minutes of sustained ischemia.
For the drug-treated groups described below, the experiments were conducted in a manner identical to those described above for the untreated control and three-cycle IP hearts.
Inhibition of
PKC
We treated control hearts (n=8) and hearts subjected to
three
cycles of IP (n=7) with a bolus dose of the PKC inhibitor
polymyxin B (Sigma Chemical Co) diluted in normal saline (24 mg/kg IV)
and administered 5 minutes before a 10-minute period of sustained
ischemia, at which time hearts were harvested for determination
of cAMP. Recently, we have confirmed that this dose of polymyxin B
administered 5 minutes before 30-minute ischemia and 90-minute
reperfusion blocks the protective effect of IP on necrosis in our in
vivo regional ischemia/reperfusion rabbit model (authors'
unpublished data, 1995).
Responsiveness of the
ß-Adrenergic Effector Pathway
We infused control hearts and hearts
subjected to three cycles
of IP with the ß-adrenergic receptor agonist isoproterenol
(diluted in normal saline [5 µg/kg IV for 5 minutes]) at the end
of
the 75-minute stabilization period (control group) and after three
cycles of transient ischemia and reperfusion (IP group) (n=7
per group). After isoproterenol infusion, we harvested the hearts for
determination of cAMP. In both groups, these cAMP measurements were
made in the myocardial region supplied by the left main
coronary artery (ie, the reperfused myocardium in
IP hearts and virgin myocardium in control hearts) and also
outside this region.
Blockade of the ß-Adrenergic
Receptor
We treated control hearts (n=5) and hearts subjected
to three
cycles IP (n=4) with a bolus dose of the ß-adrenergic receptor
blocker propranolol (diluted in normal saline [0.75 mg/kg
IV]) administered 9 minutes before a 10-minute period of sustained
ischemia, at which time hearts were harvested for determination
of cAMP.
All hearts were immersed in liquid nitrogen immediately after harvesting. We chose to harvest hearts subjected to one cycle of IP and hearts treated with polymyxin B and propranolol at 10 minutes of ischemia because the variability in cAMP measurements within the untreated control and three-cycle IP groups to which these groups were to be compared was lowest at this time point and, consequently, the statistical power was highest.
In experiments involving ischemia or ischemia and reperfusion, we delineated the area at risk from the remaining myocardium during the in vivo experiment immediately after inducing ischemia. We did this by inserting four 2-0 polypropylene suture threads onto the epicardial surface of the heart at the borderline between the area at risk and the myocardium outside this area. After coronary artery occlusion, this borderline is easily distinguishable, because there is a sharp cutoff between the cyanotic area at risk and the normally perfused myocardium adjacent to this area. For parallelism, we placed the sutures identically in the myocardial perfusion territory supplied by the left main coronary artery in hearts that were never subjected to ischemia (ie, true baseline hearts and nonischemic hearts treated with isoproterenol).
cAMP Assay
We freeze-dried the hearts and then used the polypropylene
sutures as a guide to obtain a section of 
100 mg of
myocardium from within the center of the area at risk (or
myocardial perfusion territory of the left main coronary artery
in the case of hearts that had never been subjected to
ischemia). A similar volume of tissue was obtained from virgin
left ventricular myocardium remote from this
region. cAMP determinations were made in both of these regions in all
hearts. Great care was taken to avoid tissue sampling from the border
zone.
We then pulverized and weighed the tissues and homogenized them in 6% trichloroacetic acid by using a Polytron PT 10/35 (setting at 7) for three 10-second bursts while keeping the homogenate on ice. We then centrifuged the homogenate at 1000g for 10 minutes at 4°C. We collected the supernatant and washed it in ether at five times the supernatant volume. This ether-washing step was repeated four times. The supernatant was then quick-frozen in liquid nitrogen, freeze-dried, and stored at -70°C until the time of assay. The radioimmunoassay procedure used for cAMP determination was similar to that of Steiner et al,18 as supplied by Amersham Diagnostics. In initial experiments, it was determined that recovery of [3H]cAMP (Sigma) in the freeze-dried supernatant was 85.9±0.8%. Values were not corrected for recovery, since there was little variability between measurements. We assayed all samples in duplicate; the coefficient of variation for the duplicate measurements was 5.1%. All cAMP assays were performed in a blinded fashion. cAMP measurements are expressed in nanomoles per gram dry weight.
Necrosis Determination Study
Experimental Design
All experiments were of 3-hour and 15-minute duration and began
with a 30-minute stabilization period following surgery. After the
stabilization period, hearts in the control groups received an
additional 45-minute stabilization period followed by 30 minutes of
regional ischemia and 90 minutes of reperfusion. After the
30-minute stabilization period, hearts in the IP groups received three
cycles of 5-minute ischemia and 10-minute reperfusion before
30-minute ischemia and 90-minute reperfusion.
We studied four groups of rabbits. Groups 1 (n=10) and 2 (n=13) were the untreated control and IP groups, respectively, the protocols for which are described above. Groups 3 (n=10) and 4 (n=7) were the NKH477-treated control and IP groups, respectively. The protocol for these NKH477-treated groups was identical to that for the untreated control and IP groups, except that these animals were given 45 µg/kg of NKH477 diluted in normal sterile saline 5 minutes before the 30-minute ischemia. We infused NKH477 directly into the left atrium over a 3-minute infusion period. To confirm that this dose of NKH477 increased cAMP levels within the area at risk during the 30-minute period of ischemia, we prepared rabbits in a manner identical to the control+NKH477 and IP+NKH477 groups and harvested the hearts at 10, 20, and 30 minutes of sustained ischemia (three hearts per time point per group) for determination of cAMP.
Infarct and Area-at-Risk Determination
At the end
of the experiment, we excised the hearts, mounted
them quickly onto a Langendorff apparatus, and perfused
them with normal saline to wash out the blood. We then resnared the
left main coronary artery at the in vivo occlusion site and
subsequently perfused the heart with fluorescent particles
(Duke Scientific) suspended in normal saline at a perfusion pressure of
75 mm Hg. With this technique, the fluorescent particles lodge
in all myocardium that is not part of the area at risk,
while myocardium within the area at risk remains free of
particles. After delineation of the area at risk, we removed the hearts
from the perfusion apparatus and sliced the ventricles
transversely into 3-mm-thick slices. We then incubated the slices
at 37°C for 10 minutes in a 1.25% TTC solution made up with 0.2
mol/L Tris buffer (pH 7.4). TTC stains viable myocardium
brick red, whereas areas of necrosis appear a lighter tan or brown
color. After TTC staining, we placed transparent sheets of acetate over
each slice and traced its outline, as well as the outlines of the
necrotic areas, onto the acetates. We then examined the slices under
ultraviolet light to see the fluorescent particles, and the
region of nonfluorescence (area at risk) was also traced
onto the acetates. These areas were digitized with a digitizing tablet
interfaced with a personal computer (with an Intel 486 microprocessor)
and analyzed using SIGMA SCAN software
(Jandel Scientific).
Statistical Analysis
All cAMP and hemodynamic data are
expressed as mean±SEM. In the drug-treated groups used for cAMP
determination, we evaluated hemodynamic responses to
infused drugs immediately before and 5 minutes after drug
administration by using paired t tests. We used
repeated-measures ANOVA to compare hemodynamic
variables in the four groups of the necrosis study. We
analyzed the cAMP data by using factorial ANOVA. The
Student-Newman-Keuls test was used to determine significant differences
between groups.
Because of the large reductions in necrosis produced by IP in our model, several of the IP (both untreated and NKH477-treated) hearts had <5% necrosis of the area at risk. As a result, the assumptions for parametric testing (ie, normal distributions and equal variances between groups being compared) were not met; thus, standard ANOVA techniques were not used for analysis of these data. We used the Mann-Whitney rank sum test, a nonparametric test, to compare infarct sizes between groups. Since the necrosis data did not meet the assumptions for parametric testing, the necrosis data are described in terms of median values for each group, and box plots are used to describe the key characteristics of the distribution. To compare the incidence of fibrillation between groups, we used Fisher's exact test. Statistical significance was taken as P<.05.
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Results |
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cAMP Determination Study
Hemodynamics
There were no differences in baseline hemodynamics in any of the groups of the cAMP study (data not shown). In the drug-treated groups, there were no differences in heart rate or arterial blood pressure between the control and IP groups, either before or after drug administration for any of the drugs used. Therefore, these values were pooled for statistical analysis of drug effects. Isoproterenol increased the heart rate by 18±4% of the initial value (P<.001) and reduced the mean arterial blood pressure by 17±3% (P<.01). Propranolol reduced the heart rate by 17±2% (P<.0001) and also produced an 8±3% reduction in the mean arterial pressure (P<.05). Polymyxin B administration reduced the heart rate by 12±1% (P<.0001) and produced profound hypotension, reducing the blood pressure by 50±3% of the initial value (P<.0001).
cAMP Levels in Control and IP Hearts
All cAMP
levels presented in Figs 1 to 5 show the levels
present within the area at risk in all hearts that were subjected
to ischemia or ischemia and reperfusion. In hearts that
had never been made ischemic (ie, true baseline hearts and
control hearts treated with isoproterenol), the cAMP levels in the
myocardial perfusion territory of the left main coronary artery
are given (ie, the area usually occupied by the area at risk in
experiments involving ischemia).
As shown in Fig 1
,
control hearts underwent a large
increase in cAMP levels within the area at risk at 10 minutes of
regional ischemia, and this increase persisted throughout the
30-minute ischemic period. In contrast to the substantial
increases in cAMP observed in control hearts, cAMP levels in hearts
subjected to three cycles of IP were not significantly higher than
baseline (nonischemic) values at 10, 20, or 30 minutes of
ischemia. Immediately before sustained ischemia, hearts
subjected to three cycles of IP had levels of cAMP that were modestly
but significantly lower than the levels in control hearts (3.1±0.2
nmol/g dry wt in IP hearts versus 3.9±0.2 nmol/g dry wt in control
hearts, P<.01). There were no differences in cAMP levels
between control and IP hearts in myocardium remote from the
area at risk either before or at any time during the 30-minute
sustained ischemia.
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P<.05 compared with
the baseline value.cAMP levels measured after 5-minute ischemia (which corresponds to both the end of the first 5 minutes of transient ischemia in IP hearts and 5 minutes of sustained ischemia in control hearts) measured 4.4±0.2 nmol/g dry wt. In IP hearts, cAMP levels measured at the end of the third 5-minute transient ischemia measured 4.4±0.4 nmol/g dry wt. The pooled cAMP levels measured at these two time points were significantly higher than in true baseline hearts (P<.05).
As shown in Fig
2, IP induced with only a single cycle
of 5-minute transient ischemia and 10-minute reperfusion also
reduced cAMP levels during a subsequent 10-minute period of sustained
ischemia (P<.001 versus control). However, cAMP
levels in hearts preconditioned with only a single cycle of IP were
still significantly higher than those levels measured at this time
point in hearts subjected to three cycles of IP (P<.05
versus three cycles of IP).
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Inhibition of PKC
As
shown in Fig 3, the administration of the
PKC inhibitor polymyxin B tended to lessen the increase in
cAMP that occurred at 10 minutes of regional ischemia in
control hearts. This trend did not, however, achieve statistical
significance at the .05 level (P=.10). Despite this effect,
polymyxin B–treated IP hearts still had significantly lower levels of
cAMP compared with the polymyxin B–treated control hearts
(P<.05). Polymyxin B–treated IP hearts also did not have
cAMP levels that were different from untreated IP hearts
(P=NS). In the myocardium outside the area at
risk, cAMP levels in the polymyxin B–treated control and IP hearts
were not different from each other (4.7±0.5 in control+polymyxin
B–treated hearts versus 4.1±0.5 nmol/g dry wt in
IP+polymyxin
B–treated hearts), nor were they different from the
myocardium outside the area at risk in the untreated
control and IP hearts at 10 minutes of ischemia (4.3±0.3 in
untreated control hearts versus 3.7±0.2 nmol/g dry wt in untreated
three-cycle IP hearts).
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Responsiveness of the
ß-Adrenergic Effector Pathway
As shown in Fig 4, the
ß-adrenergic receptor
agonist isoproterenol significantly increased cAMP levels in the region
previously made ischemic by three cycles of transient
ischemia and reperfusion and also in the same myocardial
territory of control hearts that were never ischemic. However,
there was no difference in the cAMP response to isoproterenol between
the two groups. Furthermore, when the cAMP response to isoproterenol in
the area previously made ischemic by three cycles of IP was
compared with myocardium outside this area in the same
heart, no difference was detected (7.0±1.2 nmol/g dry wt in the area
previously made ischemic versus 7.5±0.9 nmol/g dry wt in
nonischemic myocardium). It is interesting to
note that the cAMP levels observed within the area at risk at 10
minutes of ischemia in control hearts (9.5±0.8 nmol/g dry wt)
was significantly higher than cAMP levels achieved with this high dose
of isoproterenol in the pooled control and IP hearts (7.3±0.7 nmol/g
dry wt, P=.05).
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Blockade of the
ß-Adrenergic Receptor
As shown in Fig 5,
administration of the
ß-adrenergic receptor blocker propranolol completely
abolished the increase in cAMP levels that occurred at 10 minutes of
regional ischemia in control hearts but did not reduce cAMP
levels in IP hearts. In the myocardium outside the area at
risk, cAMP levels in propranolol-treated control and IP
hearts were not different from each other (3.7±0.3 in
control+propranolol-treated hearts versus 3.8±0.4
nmol/g dry wt in IP+propranolol-treated hearts), nor
were they different from the myocardium outside the area at
risk in untreated control and IP hearts at 10 minutes of
ischemia (4.3±0.3 in untreated control hearts versus 3.7±0.2
nmol/g dry wt in untreated three-cycle IP hearts).
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Necrosis Determination Study
The hemodynamics recorded in the
necrosis
study are presented in the Table. NKH477
significantly increased heart rate at 15 and 30 minutes of sustained
ischemia in both the control and IP groups. End-reperfusion
hemodynamics were not different among these four
groups. Fig 6 shows the infarct size expressed as a
percentage of the area at risk in all groups. The median value for
infarct size in the untreated control group was 61.9% of the area at
risk. Three cycles of transient ischemia and reperfusion
significantly protected the heart from infarction: the median necrosis
value was only 0.3% of the area at risk (P<.0001 using the
Mann-Whitney rank sum test). IP induced with three cycles of transient
ischemia and reperfusion was also highly effective in reducing
necrosis in the presence of NKH477 (median necrosis, 0.6% of the area
at risk in IP+NKH477-treated group versus 62.9% of the area at risk in
control+NKH477-treated group, P<.05). There was no
detectable difference in infarct size between the untreated control and
control+NKH477-treated groups.
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and 
) show the infarct size for each
member of the group. Note that the rectangular boxes do not
represent the mean±SEM but, rather, box plots for the group.
The rectangular box encloses 50% of the data points, the bars
extending from the box represent the remaining data points, and
the line within the box indicates the median value for the group.
Infarct sizes expressed as mean±SEM for the four groups in this necrosis study were as follows: 53.6±5.5% (of the area at risk) in the untreated control group, 3.2±1.3% in the untreated IP group, 50.2±7.7% in control+NKH477-treated group, and 10.0±5.9% in the IP+NKH477-treated group. There were no significant differences in ventricular area or area at risk between any of the four groups of this study.
Before initiating the infarct-size study, we
confirmed that the
dose of NKH477 used (45 µg/kg) increased cAMP levels within the area
at risk during the 30-minute period of regional ischemia in
both the control and IP groups. Control and IP hearts treated with
NKH477 were harvested for cAMP measurements at 10, 20, and 30 minutes
of ischemia. As shown in Fig 7, average cAMP
levels (obtained by averaging cAMP levels measured at these three
ischemic time points) were higher in the area at risk in IP
hearts treated with NKH477 than in untreated IP hearts
(P<.05) but were not different from the untreated control
hearts. NKH477 also significantly increased cAMP levels in the
control+NKH477-treated group (P<.05 versus untreated
control group). There was no difference between control and IP hearts
in the percentage increase in cAMP by NKH477 during ischemia
(66±34% in control hearts and 96±24% in IP hearts,
P=NS).
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Incidence of Fibrillation
In the untreated control and IP
groups, 1 of 10 and 2 of 13
rabbits, respectively, developed ventricular fibrillation
during sustained ischemia. All 3 of these rabbits were
successfully defibrillated after one attempt. NKH477 significantly
increased the incidence of ventricular fibrillation in the
control group (7 of 10 or 70% of the rabbits fibrillated,
P<.05 versus untreated control group by Fisher's exact
test) and also in the IP+NKH477-treated group (4 of 7 or 57% of the
rabbits fibrillated, P<.05 versus untreated IP group).
There was no significant difference in the incidence of fibrillation
between the NKH477-treated control and NKH477-treated IP groups.
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Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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The most important observations of the present study were as follows: (1) cAMP levels were increased nearly twofold during regional myocardial ischemia (ie, <30 minutes) in rabbit hearts. (2) This increase in cAMP levels was attenuated when ischemia was preceded by a single cycle of transient ischemia and reperfusion and prevented when ischemia was preceded by three cycles of transient ischemia and reperfusion. (3) Inhibition of PKC with polymyxin B did not block the cAMP-lowering effect of IP. (4) Stimulation of the ß-adrenergic receptor with isoproterenol produced similar increases in cAMP in nonischemic myocardium and in myocardium subjected to three cycles of transient ischemia and reperfusion. (5) Blockade of the ß-adrenergic receptor with propranolol abolished the increase in cAMP that occurred with sustained ischemia in control hearts but did not reduce cAMP levels in IP hearts. (6) Activation of adenylyl cyclase with NKH477 increased cAMP levels during sustained ischemia but did not increase necrosis in control hearts or block the protection of IP against necrosis.
Possible Mechanisms of Reduced cAMP in IP
Activation of
PKC
Evidence has emerged suggesting that activation of PKC is the
final effector pathway responsible for the protection of IP against
necrosis3 and postischemic
dysfunction.4 Activation of PKC is also known to produce
several effects on the cAMP-generating and -degrading pathways.
However, the net effect of PKC activation on myocardial cAMP levels is
difficult to predict from available studies, because one of these
effects would be expected to decrease cAMP (eg, uncoupling of adenylyl
cyclase–coupled receptors from Gs protein), whereas
others would be expected to increase cAMP (eg, ablation of
Gi function19 and direct sensitization of
adenylyl cyclase20 ). In the present study, we
demonstrated that the PKC inhibitor polymyxin B did not
raise cAMP levels during sustained ischemia in IP hearts. This
suggests that the cAMP-lowering effect of IP is not mediated by a
PKC-dependent process. Although we found that the dose of polymyxin B
used produced a 50% reduction in mean arterial blood
pressure, we do not believe that this influenced our conclusion, since
the extent of hypotension was similar between control and IP groups.
Furthermore, polymyxin B did not affect cAMP levels in
nonischemic myocardium outside the area at
risk.
In control hearts, polymyxin B tended to blunt the rise in cAMP levels observed during sustained ischemia. One possible explanation for this effect is provided by data from Strasser et al,21 who demonstrated that in the isolated rat heart, adenylyl cyclase is itself sensitized between 5 and 20 minutes of sustained myocardial ischemia and that this sensitization was completely prevented by preperfusing these hearts with either the PKC inhibitors polymyxin B or staurosporine. Thus, in the present study, polymyxin B may have reduced the rise in cAMP in control hearts during ischemia by preventing the PKC-mediated sensitization of adenylyl cyclase.
The dose of polymyxin B used to investigate the effect of PKC on cAMP levels in the present study was selected because this dose blocks the necrosis protection of IP induced with a single cycle of 5-minute ischemia and 10-minute reperfusion in our in vivo model of regional ischemia and reperfusion (authors' unpublished data, 1995) as well as in a previous study in the rabbit.3 Although we presume that blockade of the antinecrosis effect of IP was mediated by the inhibition of PKC by polymyxin B, we must point out that we did not directly demonstrate that PKC was inhibited in our necrosis study. Therefore, it is possible that the blockade of necrosis protection by polymyxin B was mediated by a mechanism other than through its inhibitory effect on PKC.
Reduced
Responsiveness of the ß-Adrenergic Effector
Pathway
Our results show that control and IP hearts had a similar
increase
in cAMP in response to the ß-adrenergic receptor agonist
isoproterenol and thus argue against our hypothesis that the
ß-adrenergic effector pathway is desensitized by transient
ischemia and reperfusion. Further evidence against
ß-adrenergic receptor desensitization in IP comes from the work
of Iwase et al,22 who compared the changes in myocardial
ß-adrenergic receptor number and the functional activities of
Gs and adenylyl cyclase during sustained ischemia
in control and IP rabbit hearts. These investigators showed that IP did
not affect the sarcolemmal density of ß-adrenergic receptors,
although it prevented the early reductions in Gs and
adenylyl cyclase activity. Furthermore, in reconstituted cell membranes
prepared from the sarcolemma of control and IP hearts that had
undergone sustained ischemia, these
investigators22 found that cAMP production in
response to isoproterenol was actually enhanced in IP hearts. Thus,
their data indicate that not only did IP not desensitize
ß-adrenergic signal transduction in ischemia, it was
found to preserve it. Thus, our findings and those of Iwase et al both
suggest that the ß-adrenergic signal transduction pathway is not
desensitized by IP.
Recently, Niroomand et al23 have reported that in canine myocardium, Gi proteins were sensitized during reperfusion following a single 5-minute period of transient ischemia and that this sensitization was maintained during a subsequent period of ischemia. Furthermore, these investigators demonstrated that this sensitization of Gi led to an increase in Gi-mediated inhibition of adenylyl cyclase in response to isoproterenol. Although enhanced inhibition of adenylyl cyclase by Gi could offer a potential explanation for the lack of elevation in cAMP levels with IP, we believe that this is unlikely. In our ß-adrenergic responsiveness experiments, the cAMP levels measured in response to isoproterenol reflect not just the cAMP production that is mediated through the ß-adrenergic receptor/Gs/adenylyl cyclase effector pathway but, rather, the summation of all influences that alter cAMP levels in response to ß-adrenergic receptor stimulation. Thus, if three cycles of transient ischemia and reperfusion had increased the activation of Gi, this effect would also have been detected as a lesser rise in cAMP levels in response to isoproterenol in IP hearts compared with nonischemic hearts. Since this was not observed, these data would argue against an important role for the activation of Gi in the cAMP-lowering effect of IP. Adenosine receptor stimulation is also not likely to be responsible for the lack of elevation in cAMP levels in IP hearts, because in pilot experiments we determined that the nonspecific adenosine receptor blocker 8-p-sulfophenyl-theophylline, given at a dose of 10 mg/kg 5 minutes before each of the three episodes of transient ischemia, also did not raise cAMP levels during sustained ischemia in IP hearts (authors' unpublished data, 1994).
Another possible explanation for the lack of elevation in cAMP levels in IP hearts is the increased activation of phosphodiesterases. However, two pieces of evidence argue against this possibility. First, as mentioned above, the cAMP levels measured in response to isoproterenol reflect the summation of all influences that alter cAMP levels in response to ß-adrenergic receptor stimulation. Therefore, if phosphodiesterase activity was increased by three cycles of transient ischemia and reperfusion, this would also have been detected as an attenuated increase in cAMP in response to isoproterenol in these IP hearts compared with nonischemic hearts, which was not observed. Second, if phosphodiesterase activity was higher in IP hearts during sustained ischemia, we would have expected IP hearts to have exhibited a lesser rise in cAMP after the administration of the adenylyl cyclase activator NKH477 compared with control hearts. However, cAMP levels during ischemia rose an average of 96% and 66% in IP and control hearts treated with NKH477, respectively. Since the increase in cAMP levels after the administration of NKH477 was clearly not reduced in IP hearts, these data also do not support a role for phosphodiesterase activation in the cAMP-lowering effect of IP.
In the present study, we investigated the cAMP response to a single high dose of isoproterenol. Although the generation of a dose-response curve using lower doses of isoproterenol may have allowed us to detect subtle differences between the control and IP groups, there was no trend toward reduced responsiveness to isoproterenol in hearts subjected to three cycles of transient ischemia and reperfusion. Therefore, we are confident of our conclusion that reduced responsiveness of the ß-adrenergic effector pathway cannot account for the lack of elevation in cAMP levels observed with IP.
Reduced Stimulation of the
ß-Adrenergic Receptor
Several lines of evidence support the
conclusion that the high
levels of cAMP we observed with sustained ischemia were caused
by increased norepinephrine release leading to increased
ß-adrenergic receptor activation and, furthermore, that the lack
of elevation in cAMP levels in IP hearts was mediated by reduced
ß-adrenergic receptor stimulation by norepinephrine
release.
With respect to the increase in cAMP levels that occurred with sustained ischemia (control hearts), our data demonstrate that ß-receptor blockade with propranolol completely abolished this increase in cAMP levels, thereby establishing that ß-adrenergic receptor activation was responsible for this rise. Previous studies have also reported that ß-adrenergic receptor blockade can either prevent24 or reduce25 ischemia-induced increases in cAMP. In addition, in rats with severely depleted myocardial norepinephrine in association with cardiac hypertrophy, increases in cAMP levels do not occur with ischemia.26
In further support of the conclusion that the increased cAMP levels we observed with myocardial ischemia were caused by increased norepinephrine release, Schömig8 has reported that periods of myocardial ischemia of >10 minutes result in extremely high extracellular concentrations of norepinephrine in the ischemic myocardium (100 to 1000 times normal plasma concentrations). This release of norepinephrine (called nonexocytotic release) does not depend on local sympathetic activation but, rather, on local metabolic factors. These factors cause the uptake1 carrier, which normally functions to reuptake norepinephrine into the sympathetic nerve terminal, to reverse its normal transport direction and produce efflux of norepinephrine from the cytoplasm of the nerve terminals to the extracellular space.8 The time frame during which this nonexocytotic norepinephrine release has been observed (between 10 and 40 minutes of ischemia) is consistent with the time frame of increased cAMP levels (between 10 and 30 minutes of ischemia) in the control hearts in the present study. Before 10 minutes of ischemia, norepinephrine release has been shown to be much less than that occurring with longer ischemic times and to occur through the normal exocytotic process.8 This observation is also consistent with the modest increase in cAMP we observed after 5 minutes of ischemia.
There are also multiple pieces of evidence suggesting that the lack of elevation in cAMP levels in IP hearts is mediated by reduced ß-adrenergic receptor activation secondary to reduced norepinephrine release. First, in contrast to the dramatic reductions in cAMP we achieved with ß-adrenergic receptor blockade in control hearts, ß-blockade had no effect on cAMP levels in IP hearts, thereby supporting the notion that ß-adrenergic receptor–mediated cAMP production was already minimal in these hearts. Since our ß-adrenergic receptor responsiveness experiments and the study by Iwase et al22 have demonstrated that the ß-adrenergic receptor signal transduction pathway is not desensitized by IP, a logical explanation for the lack of elevation in cAMP levels in IP hearts during sustained ischemia is through reduced stimulation of the ß-adrenergic receptor by reduced norepinephrine release. Furthermore, Seyfarth et al9 have recently demonstrated that IP results in dramatic reductions in norepinephrine release during sustained ischemia in the isolated rat heart. It is also interesting to note that Seyfarth et al have demonstrated a clear dose dependence, with multiple cycles of IP producing a greater attenuation of norepinephrine release during sustained ischemia compared with a single cycle of IP. We have also observed a similar dose dependence with respect to cAMP levels: three cycles of IP completely prevented the rise in cAMP that occurs with sustained ischemia, whereas a single cycle resulted in only an attenuation of the rise.
Although the mechanism responsible for the reduction of norepinephrine release during sustained ischemia by IP is not known, it should be stressed that this effect is not through depletion of norepinephrine stores by transient ischemia. In fact, Banerjee et al27 report that <3% of the total norepinephrine stores in the rat heart are released by brief periods of myocardial ischemia. Further evidence that argues against the operation of a norepinephrine depletion mechanism in IP comes from the work of Miyazaki and Zipes,28 who demonstrated that IP protects the dog heart against sympathetic denervation during the first hour of coronary occlusion and preserves sympathetic reflexes.
Although our evidence (which indicates that the lack of elevation in cAMP levels during sustained ischemia in IP hearts is mediated by a suppression of norepinephrine release) is indirect, these data taken together with previous studies by Schömig,8 Seyfarth et al,9 and Miyazaki and Zipes28 argue strongly that IP acts to protect the sympathetic nerves and either reduces or delays the nonexocytotic release of norepinephrine, which in turn prevents the intracellular elevation in cAMP that normally accompanies sustained ischemia. Our ß-receptor responsiveness data do not support the hypothesis that the cAMP-lowering effect of IP is mediated through increased inhibition of adenylyl cyclase through Gi.
Role of Low cAMP Levels in the Necrosis Protection of
IP
In untreated hearts, IP using three cycles of transient
ischemia and reperfusion resulted in a >90% reduction in
necrosis in our model, which, to our knowledge, is the greatest
reduction in necrosis ever reported with IP. The infarct
size–limiting effect of IP was still highly evident in the
presence of high levels of cAMP during sustained ischemia,
indicating that a reduction in cAMP levels was not necessary for its
protection. Significant elevations in cAMP also did not produce any
detectable increase in necrosis in control hearts, an effect that might
have been expected if these high levels of cAMP aggravated
ischemic injury.
In the present study, the adenylyl cyclase activator NKH477 was used to increase cAMP levels. NKH477 is a derivative of the drug forskolin but differs from forskolin in that it is water soluble, longer acting, and more than twice as efficacious in stimulating cardiac adenylyl cyclase activity.29 The dose of NKH477 used in the present study was selected on the basis of its ability to increase cAMP levels within the area at risk during the 30 minutes of sustained ischemia in IP hearts to levels that were similar to levels in untreated control hearts. This dose of NKH477 also significantly increased heart rate throughout the 30-minute period of ischemia, a known effect of elevated cAMP levels.
In a model identical to that used in the present study, we have previously shown that infusion of the adenylyl cyclase activator forskolin throughout the three cycles of transient ischemia and reperfusion in IP hearts also did not block the protection of IP against necrosis, even though it significantly increased cAMP levels.30 This indicates that the reduction in cAMP levels observed in IP hearts during the transient reperfusion period before the sustained ischemia was also not necessary for its protection against necrosis.
Could the Reduction in cAMP Levels in IP Be Responsible for Its
Antiarrhythmic Effect?
In addition to protecting against necrosis, IP
has also been shown
to protect against arrhythmias in several
species,31 including the conscious rabbit.32
A large body of experimental evidence also suggests that high levels of
cAMP are causally linked to the occurrence of arrhythmias and
that reducing cAMP levels with drugs such as ß-adrenergic
receptor blockers have antiarrhythmic effects.33
Therefore, the possibility exists that the absence of a rise in cAMP
levels we observed in IP hearts was causally linked to its
antiarrhythmic effect. The present study was not designed to
address the issue of arrhythmias, because anesthetized
rabbits are not prone to them in the absence of some
arrhythmia-inducing intervention. Thus, on the basis of the
infrequent occurrence of ventricular fibrillation in both
the untreated control and IP hearts, it is not possible to say whether
or not IP protected against arrhythmias in our model. However,
we did note that elevating cAMP levels with NKH477 significantly
increased the occurrence of fibrillation in both the control and IP
groups, thereby suggesting that high levels of cAMP are proarrhythmic
in our model. Interestingly, the increase in fibrillation in
NKH477-treated control hearts was not accompanied by increased
necrosis. Previous work, reported by Opie et al34 has also
demonstrated that isoproterenol, given at a dose that significantly
increases cAMP, increased fibrillation without increasing infarct size
in a porcine regional ischemia/reperfusion model.
Implicit in the proposition that IP may protect against arrhythmias through a reduction of cAMP (but that this is not involved in its protection against necrosis) is the notion that IP protects against arrhythmias and necrosis by separate specific mechanisms. This has recently been suggested by Cohen et al,32 who observed that preconditioning protocols used to produce protection against necrosis did not protect against arrhythmias and, furthermore, that protection against arrhythmias can be achieved without protection against necrosis. Further evidence suggesting that the protection of IP against arrhythmias and necrosis may be mediated by different pathways comes from the work of Speechly-Dick et al,35 who showed that in the rat, the specific PKC inhibitor chelerythrine completely blocked the protection of IP against necrosis, whereas it was unable to block the protection of IP against arrhythmias. Furthermore, these investigators found that the specific PKC agonist 1,2-dioctanoyl-sn-glycerol mimicked the protection of IP against necrosis but was unable to protect against arrhythmias.
Also noteworthy is that the protection of IP against necrosis has been suggested to be an all-or-none phenomenon, with one cycle of transient ischemia conferring similar protection to that observed with multiple cycles.36 However, Lawson and Hearse31 have shown that the protection of IP against arrhythmias shows a definite dose dependence, with a cumulative increase in protection against arrhythmias when the number of cycles used to produce IP is increased from one to three. As mentioned above, the ability of IP to suppress cAMP levels and norepinephrine release during sustained ischemia also shows a similar dose dependence. Thus, the extent to which IP protects against arrhythmias may depend on the extent to which norepinephrine release and, subsequently, myocardial intracellular cAMP levels are reduced by the transient cycles of ischemia and reperfusion.
In summary, IP prevents the rise in cAMP levels that occurs during sustained ischemia. This effect is not mediated by activation of PKC or by reduced responsiveness of the ß-adrenergic effector pathway but most likely through an attenuation of norepinephrine release. Furthermore, the antinecrotic effect of IP is not mediated by the lack of elevation in cAMP levels during sustained ischemia. However, the cAMP-lowering effect of IP may well be involved in its protection against arrhythmias. Further studies designed specifically to address the issue of arrhythmias are needed to define the precise relation between the prevention of norepinephrine release, the reduction of intracellular cAMP levels, and the antiarrhythmic effect of IP.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by Ontario Heart and Stroke Foundation grant T-2687. Dr Sandhu was the recipient of an Ontario Ministry of Health Fellowship. Dr Wilson was the recipient of a Career Investigator Award of the Ontario Heart and Stroke Foundation. We thank Dr A.K. Sen for advice. We gratefully acknowledge Nippon Kyauka Pharmaceuticals for providing NKH477.
Received June 29, 1995; accepted September 25, 1995.
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References |
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