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Experimental Research Laboratory, Division of Cardiology, University of Louisville, and Jewish Hospital Heart and Lung Institute, Louisville, Kentucky 40292
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Although Src protein tyrosine kinases (PTKs) have been shown to be essential in late preconditioning (PC) against myocardial stunning, their role in triggering versus mediating late PC against myocardial infarction remains unclear. Four groups of conscious rabbits were subjected to a 30-min coronary occlusion on day 2, with or without PC ischemia on day 1. Administration of the Src PTK inhibitor lavendustin A (LD-A; 1 mg/kg iv) before the PC ischemia on day 1 (group III, n = 7) failed to block the delayed protective effect against myocardial infarction 24 h later. Late PC against infarction, however, was completely abrogated when LD-A was given 24 h after the PC ischemia, prior to the 30-min occlusion on day 2 (group IV, n = 8). We conclude that, in conscious rabbits, Src PTK activity is necessary for the mediation of late PC protection against myocardial infarction on day 2, but not for the initiation of this phenomenon on day 1. Taken together with previous studies in the setting of stunning, these findings reveal heretofore unrecognized differences in the roles of Src PTKs in late PC against stunning versus late PC against infarction.
lavendustin A; ischemia-reperfusion injury
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE LATE PHASE OF
ISCHEMIC PRECONDITIONING (PC) is a powerful adaptive
phenomenon evoked by brief episodes of ischemia that renders
the heart resistant to subsequent ischemic stress occurring 24-72 h later (2, 4, 15). Recent evidence supports a
role of protein tyrosine kinases (PTKs) in the signaling mechanism underlying the late phase of PC (8, 9, 13, 23).
Specifically, administration of genistein (1), a rather
nonspecific inhibitor of various kinases, prior to the PC
ischemia, has been reported to block the development of late PC
against infarction in open-chest rabbits (13). In
conscious rabbits, inhibition of the Src family of PTKs with
lavendustin A (LD-A) has been found to abrogate late PC against
myocardial stunning (9). The late PC protection was
abolished both when LD-A was administered on day 1 (prior to
the PC ischemia) and when it was given on day 2 (prior to the index ischemia), suggesting a role of Src PTK
signaling both in the initiation and in the mediation of protection
(9). Consistent with this, two members of the Src family
of PTKs, Src and Lck, have been shown to be activated shortly after the
PC stimulus (on day 1) (14, 31) as well as
24 h later (on day 2) (41). In addition,
pretreatment with genistein has been shown to decrease heat shock
protein 72 expression 24 h after PC ischemia, to suppress action potential duration shortening during the index ischemia, and to abrogate late PC protection against infarction in open-chest rabbits (23). A role of PTKs in adenosine A1
receptor agonist-induced delayed protection against infarction has also
been reported (8). Recent studies in Lck
/
mice have provided unequivocal evidence for an obligatory role of this
PTK in late PC against infarction (29).
Although these studies (8, 9, 13, 14, 23, 31, 41) implicate PTKs in the signaling pathways of late PC, the role of these kinases in late PC against myocardial infarction remains unclear. In this regard, it is important to distinguish the cellular mechanisms that initiate (trigger) the development of late PC immediately after the first ischemic stress (day 1) from those that mediate cardioprotection 24-72 h later (days 2-4). While several previous studies (8, 13, 23) examined the role of PTKs in triggering late PC against infarction on day 1, the role of PTKs as mediators of cardioprotection on day 2 has not been explored. Thus it is unknown whether PTK-dependent signaling is important only to trigger or also to mediate late PC against lethal ischemia-reperfusion injury. The bifunctional role (triggers and mediators) of PTKs observed in the setting of late PC against myocardial stunning (9) cannot be extrapolated to late PC against myocardial infarction because stunning and infarction represent two different types of cellular injury. Furthermore, ischemic PC activates not only Src and Lck (31), but also protein kinase C (PKC) (30), and possibly various other kinases. Most previous studies examining the role of PTKs in late PC against infarction have used genistein (13, 23). Despite its initial description as a specific inhibitor of PTKs (1), genistein is now known to exhibit extensive nonspecific actions, including inhibition of serine-threonine kinases [e.g., PKC and protein kinase A (PKA)] (1, 11), action on multiple ion channels (22, 27), inhibition of topoisomerase II (17), and inhibition of reactive oxygen species (ROS) production in response to specific stimuli (37). Because of these potential confounding actions of genistein, it remains unclear whether the effects observed with genistein in the previously reported studies (13, 23) can be ascribed specifically to inhibition of PTK activity.
The present study was undertaken to address these unresolved issues. Using a conscious rabbit model, we tested the hypothesis that PTKs play a dual role (triggers and mediators) in the pathophysiology of late PC against myocardial infarction, i.e., that they are essential not only for the initiation of this phenomenon on day 1 but also for the manifestation of cardioprotection on day 2. Accordingly, this study had two aims. First, we determined whether administration of the PTK inhibitor LD-A prior to the first ischemic stress (on day 1) blocks the development of late PC against myocardial infarction. Second, we investigated whether administration of LD-A prior to the second ischemic stress (on day 2) abrogates the cardioprotection afforded by late PC against infarction. None of the currently available PTK inhibitors is absolutely specific. Although LD-A has been reported to inhibit phosphatidylinositol-3 kinase (25), GLUT-1 (40), and the muscarinic K+ current (26), this agent was chosen because 1) it has fewer nonspecific actions than genistein (21, 24, 25), 2) it is more selective for the Src family of PTKs than other PTK inhibitors (21, 24), and 3) it has previously been shown to inhibit Src and Lck PTKs in our conscious rabbit model of late PC (31).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study was performed in accordance with the guidelines of the Animal Care and Use Committee of the University of Louisville School of Medicine and with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, Publication No. 86-23).
Experimental preparation. The conscious rabbit model of myocardial infarction has been described in detail previously (35) and will be briefly summarized here. New Zealand White male rabbits (weight, 2.0-3.0 kg) were instrumented under sterile conditions with a balloon occluder around a major branch of the left coronary artery, a 10-MHz pulsed Doppler ultrasonic crystal in the center of the region to be rendered ischemic, and bipolar electrocardiogram (ECG) leads on the chest wall. The wires and the occluder tubing were tunneled under the skin and exteriorized through small incisions between the scapulae. The chest wound was closed in layers, and a small tube was left in the thorax for 3 days to evacuate air and fluids postoperatively. Gentamicin was administered before surgery and on the first and second postoperative days (0.5 mg/kg im each day). Rabbits were allowed to recover for a minimum of 10 days after surgery.
Experimental protocol. Throughout the experiments, rabbits were kept in a cage in a quiet, dimly lit room. Left ventricular (LV) systolic wall thickening (WTh), range gate depth, and the ECG were recorded throughout the experiments on a thermal array chart recorder (Gould TA6000; Valley View, OH). Regional myocardial function was assessed as systolic thickening fraction by use of the pulsed Doppler probe, as previously described (7). All rabbits were subjected to a 30-min coronary artery occlusion followed by 3 days of reperfusion. The performance of successful coronary occlusion was verified by observing the development of S-T segment elevation and changes in the QRS complex on the ECG and the appearance of paradoxical wall thinning on the ultrasonic crystal recordings. Diazepam was administered 20 min before the onset of ischemia (4 mg/kg ip) to relieve the stress caused by the coronary occlusion. No antiarrhythmic agents were given at any time.
The experimental protocol is illustrated in Fig. 1. Rabbits were assigned to four groups (Fig. 1). Group I (control group) underwent a 30-min occlusion with no PC protocol and no drug treatment. Group II (PC group) underwent a sequence of six 4-min coronary occlusions interspersed with 4-min intervals of reperfusion 24 h before the 30-min coronary occlusion. Group III (PC + LD-A on day 1 group) underwent the same protocol as group II except that the rabbits received an intravenous bolus of LD-A (1 mg/kg) 10 min before the six occlusion/reperfusion cycles of PC ischemia. Group IV (LD-A on day 2 group) underwent the same protocol as group III except that the rabbits received LD-A on day 2, 10 min prior to the 30-min occlusion. LD-A (Calbiochem; San Diego, CA) was dissolved under sterile conditions in 10% (vol/vol) dimethylsulfoxide (DMSO) in normal saline (total dose of DMSO, 0.1 ml/kg; total volume infused, 1 ml/kg). This dose of LD-A has been shown to completely block the activation of Src and Lck PTKs after the six 4-min occlusion/reperfusion cycles in our conscious rabbit model (31).
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Measurement of regional myocardial function. Regional myocardial function was assessed as systolic thickening fraction by use of the pulsed Doppler probe, as previously described (7). Percent systolic thickening fraction was calculated as the ratio of net systolic WTh to end-diastolic WTh, multiplied by 100 (7). The total deficit of systolic WTh over the 3-day reperfusion period (an integrative assessment of the overall severity of contractile dysfunction during this time interval) was calculated by measuring the area between the systolic WTh versus time line and the baseline (100% line) during the 3-day recovery phase after the 30-min coronary occlusion (5). In all animals, measurements were averaged from 10 beats at baseline and from 5 beats at all subsequent time points.
Measurement of region at risk and infarct size. At the conclusion of the study, the rabbits were given heparin (1,000 units iv), after which they were anesthetized with pentobarbital sodium (50 mg/kg iv) and euthanized with KCl. The heart was excised, and the size of the ischemic-reperfused region (region at risk) was determined by tying the coronary artery at the site of the previous occlusion and by perfusing the aortic root for 2 min with a 5% solution of Phthalo blue dye in normal saline at a pressure of 70 mmHg using a Langendorff apparatus. The heart was then cut into six to seven transverse slices, which were incubated for 10 min at 37°C in a 1% solution of triphenyltetrazolium chloride in phosphate buffer (pH 7.4). All atrial and right ventricular tissues were excised. The slices were weighed, fixed in a 10% neutral buffered formaldehyde solution, and photographed (Nikon AF N6006). Transparencies were projected onto a paper screen at a 10-fold magnification, and the borders of the infarcted, ischemic-reperfused, and nonischemic regions were traced. The corresponding areas were measured by computerized planimetry (Adobe Photoshop, version 4.0), and from these measurements, infarct size was calculated as a percentage of the region at risk (35).
Statistical analysis. Data are reported as means ± SE. Heart rate and thickening fraction were analyzed by a two-way repeated-measures ANOVA (time and group). Infarct sizes and risk region sizes were analyzed with a one-way ANOVA followed by Student's t-tests for unpaired data with the Bonferroni correction (32, 42). The relationship between infarct size and risk region size was compared among groups with an analysis of covariance (ANCOVA), with size of the risk region used as the covariate. The correlation between infarct size and risk region size was assessed by linear regression analysis using the least-squares method. All statistical analyses were performed using the SAS software system (32).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Exclusions. Of the 40 rabbits instrumented for this study, 11 were assigned to the control group (group I), 11 to the PC group (group II), 9 to the PC + LD-A on day 1 group (group III), and 9 to the PC + LD-A on day 2 group (group IV). Five rabbits died of ventricular fibrillation during coronary occlusion (1 in the control group, 1 in the PC group, 2 in the PC + LD-A on day 1 group, and 1 in the PC + LD-A on day 2 group). Therefore, a total of 10 rabbits completed the protocol in the control group, 10 in the PC group, 7 in the PC + LD-A on day 1 group, and 8 in the PC + LD-A on day 2 group. No rabbit included in the final analysis was subjected to defibrillation.
Hemodynamic variables.
Previous studies in conscious rabbits have shown that the dose of LD-A
used in the present investigation does not affect systemic arterial
pressure, heart rate, or systolic thickening fraction (9).
On the day of the 30-min coronary occlusion, heart rate did not differ
among the four groups at baseline (pretreatment), after treatment
(preocclusion), at 15 min into the occlusion, and at 1, 3, and 5 h
of reperfusion (Table 1).
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Region at risk and infarct size.
There were no significant differences among groups I,
II, III, and IV with respect to LV weight
(4.4 ± 0.2, 4.4 ± 0.1, 4.1 ± 0.1, and 4.5 ± 0.2 g, respectively) or weight of the region at risk [0.8 ± 0.1 g (18.2 ± 1.8% of LV weight), 0.8 ± 0.1 g
(18.0 ± 1.6% of LV weight), 0.7 ± 0.1 g (17.7 ± 2.6% of LV weight), and 0.8 ± 0.1 g (18.0 ± 2.2% of
LV weight), respectively]. The average infarct size was 44% smaller
in group II than in control animals (group I)
[30.5 ± 2.9% versus 54.2 ± 4.2% of the region at risk,
respectively; P < 0.05 (Fig.
2)], indicating a late PC effect against
myocardial infarction. In group III, infarct size (24.6 ± 6.6% of the region at risk) was also smaller than in controls and
essentially indistinguishable from the PC group (Fig. 2), indicating
that LD-A, when administered prior to the PC ischemia on
day 1, did not have any appreciable effect on the
development of late PC against infarction. In group IV,
however, infarct size (54.4 ± 3.8% of the region at risk) was
similar to that in the control group and significantly larger than that
in group II, indicating that administration of LD-A on
day 2 completely abrogated the protective effects of late PC
against infarction. In all four groups, the size of the infarction was
positively and linearly related to the size of the region at risk
(r = 0.80, 0.57, 0.84, and 0.81 in groups I,
II, III, and IV, respectively). As
expected, the regression line was significantly shifted to the right
in group II compared with group I
(P < 0.05 by ANCOVA) (Fig.
3) and group IV, indicating
that late PC reduced infarct size independent of the risk region size
and that this protective effect was abrogated by administration of LD-A
on day 2. In contrast, in group III, regression
line was very similar to that in group II and significantly different (P < 0.05 by ANCOVA) from that in
group I, indicating that for any given size of the region at
risk, the resulting infarction was smaller in preconditioned rabbits
treated with LD-A prior to the PC ischemia on day 1 than in untreated control rabbits (Fig. 3). Regression equations are
given in the legend to Fig. 3.
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Regional myocardial function.
Because of Doppler probe malfunction, complete measurements of WTh for
3 days after reperfusion could be obtained in only 8 of 10 rabbits in
group I, 8 of 10 rabbits in group II, and 4 of 8 rabbits in group IV. As expected (9), WTh
before the 30-min coronary ischemia was not altered by LD-A
(groups III and IV): systolic thickening fraction
averaged 29.0 ± 3.8% and 40.4 ± 5.3% before LD-A versus
31.7 ± 4.5% and 41.1 ± 4.8% after LD-A in groups III and IV, respectively (P = not
significant). After release of the 30-min occlusion, control rabbits
(group I) exhibited essentially no recovery of WTh even at 3 days (Fig. 4). In preconditioned rabbits
(group II), recovery of WTh was significantly
(P < 0.05) improved compared with controls at 5 h, 1 day, 2 days, and 3 days after reperfusion (Fig. 4). The total
deficit of WTh over the 3-day reperfusion period [an integrative
assessment of the overall severity of contractile dysfunction during
this time interval (5)] was decreased by 22% in
group II versus group I (P < 0.05) (Fig. 4). In group III (LD-A on day 1 group), the total deficit of WTh was 13% less than that in group
I (P < 0.05), indicating that administration of
LD-A on day 1 did not abolish the late PC protection.
Administration of LD-A on day 2, however, abrogated the
salutary actions of late PC on recovery of myocardial function in
group IV and the total deficit of WTh was similar to that in the control group (group I).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Despite identification of several key components, including nitric
oxide (NO), ROS, PKC, nuclear factor-
B, signal transducer and
activator of transcription (STAT)1/3, and prostanoids (4), the molecular mechanisms that underlie the development and mediation of
late PC remain incompletely understood. Recent studies have shown that
ischemic PC activates two members of the Src family of PTKs,
Src and Lck, in the rabbit heart (31), that PTKs are involved in both the initiation and in the mediation of the delayed protection against myocardial stunning via upregulation of NO synthesis
in rabbits (9), and that Lck is necessary for the initiation and/or mediation of late PC against infarction in mice (29). The role of PTKs in the initiation versus mediation
of late PC against infarction, however, is unclear. Furthermore, nonuniform results have been reported concerning the function of
PTKs in early PC against myocardial infarction (10, 14, 18, 19,
38).
Salient findings. Utilizing a conscious rabbit model, we found that administration of LD-A prior to the PC ischemia (on day 1), at a dose that blocks Src/Lck activation (31), failed to block the protective effects of late PC against myocardial infarction, indicating that Src PTK activity is not necessary for the development of this phenomenon. In contrast, the infarct-sparing effects of late PC were completely abrogated when LD-A was administered on day 2, indicating a pivotal role of Src PTKs in the mediation of protection. To our knowledge, this is the first study to demonstrate that PTKs are involved in the mediation (on day 2) as opposed to the initiation (on day 1) of delayed protection against myocardial infarction, a finding which reveals a new role of PTKs in late PC.
PTK activity and development of late PC against infarction (day 1). Our data indicate that, although Src PTK activity is increased soon after the PC stimulus (31), it is not essential for the development (as trigger) of delayed protection against infarction. In contrast, Src PTK activity is necessary for the development of late PC against myocardial stunning in conscious rabbits (9). Specifically, the initiation of this phenomenon was abrogated by administration of LD-A (at the same dose as that used in the present investigation) prior to the PC ischemia. This dose of LD-A also abolished the late PC-induced increase in inducible NO synthase (iNOS) activity 24 h later, indicating a role of Src PTK signaling in transcriptional modulation of iNOS (9). The present finding that LD-A, administered on day 1, failed to block the infarct-sparing effects of late PC indicates that the signaling mechanism responsible for the delayed protection against infarction can be initiated without involvement of Src PTKs. The difference between these results and those obtained previously in the setting of late PC against stunning (9) can be rationalized when one considers that myocardial stunning and infarction represent two very different types of injury, and that at least under certain experimental conditions, different signaling components may be involved in the PC protection against reversible and irreversible ischemic injury (3). It must be stressed that the dose of L-DA used in the present study has been shown to completely block the activation of Src PTKs associated with ischemic PC in this same conscious rabbit model (31). Therefore, the inability of L-DA to block late PC in the present study cannot be ascribed to lack of effectiveness of the inhibitor.
The fact that inhibition of Src PTKs by LD-A blocks protection against infarction but not protection against stunning is congruent with the emerging notion that late PC is a polygenic phenomenon with multiple, often overlapping and/or redundant signaling and effector pathways (4). Besides PKC-
and Src PTKs, recent evidence indicates that other stress-responsive pathways (e.g., the Janus kinase-STAT cascade) (43) are involved in this
phenotypic shift. It is conceivable that the precise pathways recruited
during the development of late PC may vary depending upon the stimulus
(e.g., ischemia versus pharmacological or physical stimuli) and
the end point (infarction versus stunning) (4). Important
mechanistic differences among different forms of late PC have already
been demonstrated, such as the fact that ATP-sensitive K+
(KATP) channels play an essential role in late PC against
infarction but not in late PC against stunning (36), that
increased NOS activity is essential for adenosine A1
receptor-induced but not adenosine A3 receptor-induced late
PC (34), and that activation of adenosine A1
receptors induces late PC against infarction (34) but not
against stunning (16).
We have recently found that genetic ablation of Lck abrogates the
infarct-sparing effects of ischemia-induced late PC in
mice (29). Apart from the obvious species difference, this
result is not in contrast with our present data in rabbits, since it is
plausible that Lck plays an obligatory role in the mediation (as
opposed to the initiation) of late PC in both mice and rabbits. Targeted deletion of Lck does not enable one to discern whether Lck is
acting as a trigger of late PC (on day 1) or as a mediator (on day 2). We propose that the loss of protection in
Lck/ mice (29) indicates that Lck activity
is indispensable to mediate cardioprotection 24 h after the
ischemic PC stimulus, not to trigger the development of the PC
phenotype immediately after the ischemic PC stimulus, which
would be consistent with our findings in rabbits.
Our observations seem to be at variance with those of three previous
studies (8, 13, 23) that have suggested a role of PTKs in
the initiation of late PC against infarction. Two of these studies
(13, 23) used genistein to achieve broad-spectrum PTK
inhibition, whereas another study (8) interrogated the mechanism of adenosine A1 receptor-induced late PC. The
apparent discrepancy between these studies and our current observations could be the result of several factors. First, genistein has numerous nonspecific actions, including inhibition of PKC and PKA
(1), inhibition of topoisomerase II (17),
inhibition of ROS generation (37), and effects on several
ion channels in various tissues and cell lines (22, 27).
Thus genistein may potentially interact with several other key elements
of late PC signaling, including ROS (37), PKC (1,
11), and the KATP channel (22). In the
present study, the use of LD-A, an inhibitor of PTKs with fewer
nonspecific (i.e., PTK unrelated) actions (21, 24), minimized interactions with other cellular kinases. Second, at least in
early PC signaling, contradictory findings regarding the role of PTKs
have been observed when experiments were carried out in different
animal species and under different experimental conditions (10,
14, 18). The use of conscious animals (as in the present study)
eliminates the confounding effects of factors associated with
open-chest preparations, such as anesthesia, surgical trauma,
fluctuations in temperature, elevated catecholamines, excessive free
radical formation, and release of cytokines. These factors may
potentially interfere with PTK activity, PC signaling, myocardial
infarct size, or a combination of these (12, 33). Third,
at least several hundred PTKs are known to exist (20), and
it is possible that non-Src PTKs may also be involved in late PC. In
the present study the use of LD-A, a selective inhibitor of the Src and
epidermal growth factor (EGF) receptor subfamilies of PTKs
(21, 24), enabled us to interrogate these enzymes but did
not provide information regarding the potential involvement of other
PTKs. Thus the possibility that adenosine A1
receptor-induced late PC (8) may signal through subset(s)
of PTKs different from those recruited by ischemia and with
different susceptibility to inhibitors cannot be ruled out. Finally,
the sheer number of PTKs (20, 39), their widely different
susceptibilities to various classes of inhibitors, and the plethora of
known and unknown signaling functions of this superfamily of enzymes
make it difficult, if not impossible, to generalize any finding derived
from the use of a single agent with a rather narrow spectrum of
inhibition. As mentioned above, LD-A targets the Src and EGF receptor
subfamilies of PTKs (21, 24) but fails to inhibit many
other PTKs whose roles in the setting of late PC signal transduction
have not yet been tested. On the basis of the observations reported
herein, the involvement of LD-A-insensitive but genistein-sensitive
PTKs in the proximal signaling events of late PC against infarction cannot be excluded.
PTK activity and mediation of protection (day 2). A novel finding of the present study was that LD-A abolished the delayed infarct-sparing effects of late PC when administered on day 2, prior to the 30-min coronary occlusion. To our knowledge, this is the first evidence that PTK activity is essential for the mediation (as opposed to the initiation) of late PC protection against infarction. Although our findings were obtained in the setting of ischemic PC, they may help to elucidate the functional significance of the increase in Src kinase activity observed 24 h after diethylenetriamine/nitric oxide-induced PC (41). The mechanism whereby PTKs participate in the mediation of late PC remains to be ascertained. Since iNOS plays an obligatory role in the manifestation of delayed protection against both myocardial stunning and infarction (6, 35) and since administration of L-DA on day 2, prior to the second ischemic challenge, inhibits the increased iNOS activity associated with late PC (9), a plausible explanation for our findings is that PTK activity on day 2 leads to cardioprotection by augmenting iNOS activity via posttranslational modulation of iNOS by tyrosine phosphorylation. This concept is consistent with the fact that tyrosine phosphorylation of iNOS is associated with increased iNOS activity in murine macrophages (28). However, one cannot exclude the possibility that tyrosine phosphorylation and consequent activation of other mediators of late PC (e.g., cyclooxygenase-2) may also contribute. Further studies will be necessary to identify the exact signaling events governed by PTKs on day 2.
In conclusion, this study expands our understanding of the signaling infrastructure responsible for late PC against infarction by demonstrating that, in conscious rabbits, Src PTK activity is essential for this cardioprotective phenotype to become manifest 24 h after the PC stimulus (on day 2). Somewhat surprisingly, Src PTK activity does not appear to be required for the initiation of this delayed cardioprotective adaptation on day 1, a finding that contrasts with the previous demonstration that Src PTK activity is necessary for the initiation of ischemia-induced late PC against myocardial stunning (9). The present data imply that the mechanisms responsible for late PC against stunning and infarction are different, which adds to previous evidence pointing in this direction (4, 16, 36).| |
ACKNOWLEDGEMENTS |
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We gratefully acknowledge Gregg Shirk and Wen-Jian Wu for expert technical assistance and Marcia Joines and Carla Hilse for expert secretarial assistance.
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FOOTNOTES |
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This study was supported in part by National Heart, Lung, and Blood Institute Grants R01 HL-43151, HL-55757, and HL-68088 (to R. Bolli), National American Heart Association Scientist Development Grant 0130146N (to B. Dawn), the Medical Research Grant Program of the Jewish Hospital Foundation, Louisville, KY, and the Commonwealth of Kentucky Research Challenge Trust Fund.
Address for reprint requests and other correspondence: R. Bolli, Division of Cardiology, Univ. of Louisville, Louisville, KY 40292 (E-mail: rbolli{at}louisville.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpheart.00873.2001
Received 7 October 2001; accepted in final form 18 March 2002.
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REFERENCES |
|---|
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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|---|
1.
Akiyama, T,
Ishida J,
Nakagawa S,
Ogawara H,
Watanabe S,
Itoh N,
Shibuya M,
and
Fukami Y.
Genistein, a specific inhibitor of tyrosine-specific protein kinases.
J Biol Chem
262:
5592-5595,
1987
2.
Baxter, GF,
and
Ferdinandy P.
Delayed preconditioning of myocardium: current perspectives.
Basic Res Cardiol
96:
329-344,
2001[ISI][Medline].
3.
Bolli, R.
The early and late phases of preconditioning against myocardial stunning and the essential role of oxyradicals in the late phase: an overview.
Basic Res Cardiol
91:
57-63,
1996[ISI][Medline].
4.
Bolli, R.
The late phase of preconditioning.
Circ Res
87:
972-983,
2000
5.
Bolli, R,
Bhatti ZA,
Tang XL,
Qiu Y,
Zhang Q,
Guo Y,
and
Jadoon AK.
Evidence that late preconditioning against myocardial stunning in conscious rabbits is triggered by the generation of nitric oxide.
Circ Res
81:
42-52,
1997
6.
Bolli, R,
Manchikalapudi S,
Tang XL,
Takano H,
Qiu Y,
Guo Y,
Zhang Q,
and
Jadoon AK.
The protective effect of late preconditioning against myocardial stunning in conscious rabbits is mediated by nitric oxide synthase. Evidence that nitric oxide acts both as a trigger and as a mediator of the late phase of ischemic preconditioning.
Circ Res
81:
1094-1107,
1997
7.
Bolli, R,
Zhu WX,
Myers ML,
Hartley CJ,
and
Roberts R.
Beta-adrenergic stimulation reverses postischemic myocardial dysfunction without producing subsequent functional deterioration.
Am J Cardiol
56:
964-968,
1985[ISI][Medline].
8.
Dana, A,
Skarli M,
Papakrivopoulou J,
and
Yellon DM.
Adenosine A(1) receptor induced delayed preconditioning in rabbits: induction of p38 mitogen-activated protein kinase activation and Hsp27 phosphorylation via a tyrosine kinase- and protein kinase C-dependent mechanism.
Circ Res
86:
989-997,
2000
9.
Dawn, B,
Xuan YT,
Qiu Y,
Takano H,
Tang XL,
Ping P,
Banerjee S,
Hill M,
and
Bolli R.
Bifunctional role of protein tyrosine kinases in late preconditioning against myocardial stunning in conscious rabbits.
Circ Res
85:
1154-1163,
1999
10.
Fryer, RM,
Schultz JE,
Hsu AK,
and
Gross GJ.
Pretreatment with tyrosine kinase inhibitors partially attenuates ischemic preconditioning in rat hearts.
Am J Physiol Heart Circ Physiol
275:
H2009-H2015,
1998
11.
Geissler, JF,
Traxler P,
Regenass U,
Murray BJ,
Roesel JL,
Meyer T,
McGlynn E,
Storni A,
and
Lydon NB.
Thiazolidine-diones Biochemical and biological activity of a novel class of tyrosine protein kinase inhibitors.
J Biol Chem
265:
22255-22261,
1990
12.
Haessler, R,
Kuzume K,
Chien GL,
Wolff RA,
Davis RF,
and
Van Winkle DM.
Anaesthetics alter the magnitude of infarct limitation by ischaemic preconditioning.
Cardiovasc Res
28:
1574-1580,
1994[ISI][Medline].
13.
Imagawa, J,
Baxter GF,
and
Yellon DM.
Genistein, a tyrosine kinase inhibitor, blocks the "second window of protection" 48 h after ischemic preconditioning in the rabbit.
J Mol Cell Cardiol
29:
1885-1893,
1997[ISI][Medline].
14.
Kitakaze, M,
Node K,
Asanuma H,
Takashima S,
Sakata Y,
Asakura M,
Sanada S,
Shinozaki Y,
Mori H,
Kuzuya T,
and
Hori M.
Protein tyrosine kinase is not involved in the infarct size-limiting effect of ischemic preconditioning in canine hearts.
Circ Res
87:
303-308,
2000
15.
Kuzuya, T,
Hoshida S,
Yamashita N,
Fuji H,
Oe H,
Hori M,
Kamada T,
and
Tada M.
Delayed effects of sublethal ischemia on the acquisition of tolerance to ischemia.
Circ Res
72:
1293-1299,
1993
16.
Maldonado, C,
Qiu Y,
Tang XL,
Cohen MV,
Auchampach J,
and
Bolli R.
Role of adenosine receptors in late preconditioning against myocardial stunning in conscious rabbits.
Am J Physiol Heart Circ Physiol
273:
H1324-H1332,
1997
17.
Markovits, J,
Linassier C,
Fosse P,
Couprie J,
Pierre J,
Jacquemin-Sablon A,
Saucier JM,
Le Pecq JB,
and
Larsen AK.
Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II.
Cancer Res
49:
5111-5117,
1989
18.
Maulik, N,
Sato M,
Price BD,
and
Das DK.
An essential role of NFkappaB in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia.
FEBS Lett
429:
365-369,
1998[ISI][Medline].
19.
Maulik, N,
Yoshida T,
Zu YL,
Sato M,
Banerjee A,
and
Das DK.
Ischemic preconditioning triggers tyrosine kinase signaling: a potential role for MAPKAP kinase 2.
Am J Physiol Heart Circ Physiol
275:
H1857-H1864,
1998
20.
Neet, K,
and
Hunter T.
Vertebrate non-receptor protein-tyrosine kinase families.
Genes Cells
1:
147-169,
1996[Abstract].
21.
O'Dell, TJ,
Kandel ER,
and
Grant SG.
Long-term potentiation in the hippocampus is blocked by tyrosine kinase inhibitors.
Nature
353:
558-560,
1991[Medline].
22.
Ogata, R,
Kitamura K,
Ito Y,
and
Nakano H.
Inhibitory effects of genistein on ATP-sensitive K+ channels in rabbit portal vein smooth muscle.
Br J Pharmacol
122:
1395-1404,
1997[ISI][Medline].
23.
Okubo, S,
Bernardo NL,
Elliott GT,
Hess ML,
and
Kukreja RC.
Tyrosine kinase signaling in action potential shortening and expression of HSP72 in late preconditioning.
Am J Physiol Heart Circ Physiol
279:
H2269-H2276,
2000
24.
Onoda, T,
Iinuma H,
Sasaki Y,
Hamada M,
Isshiki K,
Naganawa H,
Takeuchi T,
Tatsuta K,
and
Umezawa K.
Isolation of a novel tyrosine kinase inhibitor, lavendustin A, from Streptomyces griseolavendus.
J Nat Prod
52:
1252-1257,
1989[Medline].
25.
Onoda, T,
Isshiki K,
Takeuchi T,
Tatsuta K,
and
Umezawa K.
Inhibition of tyrosine kinase and epidermal growth factor receptor internalization by lavendustin A methyl ester in cultured A431 cells.
Drugs Exp Clin Res
16:
249-253,
1990[ISI][Medline].
26.
Otero, A,
and
Sweitzer NM.
Benzoquinoid tyrosine kinase inhibitors are potent blockers of cardiac muscarinic receptor function.
Mol Pharmacol
44:
595-604,
1993[Abstract].
27.
Paillart, C,
Carlier E,
Guedin D,
Dargent B,
and
Couraud F.
Direct block of voltage-sensitive sodium channels by genistein, a tyrosine kinase inhibitor.
J Pharmacol Exp Ther
280:
521-526,
1997
28.
Pan, J,
Burgher KL,
Szczepanik AM,
and
Ringheim GE.
Tyrosine phosphorylation of inducible nitric oxide synthase: implications for potential post-translational regulation.
Biochem J
314:
889-894,
1996[ISI][Medline].
29.
Ping, P,
Song C,
Zhang J,
Guo Y,
Cao X,
Li R,
Wu W,
Vondriska TM,
Pass JM,
Tang XL,
Pierce WM,
and
Bolli R.
Formation of protein kinase C-Lck signaling modules confers cardioprotection.
J Clin Invest
109:
499-507,
2002[ISI][Medline].
30.
Ping, P,
Zhang J,
Qiu Y,
Tang XL,
Manchikalapudi S,
Cao X,
and
Bolli R.
Ischemic preconditioning induces selective translocation of protein kinase C isoforms epsilon and eta in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity.
Circ Res
81:
404-414,
1997
31.
Ping, P,
Zhang J,
Zheng YT,
Li RC,
Dawn B,
Tang XL,
Takano H,
Balafanova Z,
and
Bolli R.
Demonstration of selective protein kinase C-dependent activation of Src and Lck tyrosine kinases during ischemic preconditioning in conscious rabbits.
Circ Res
85:
542-550,
1999
32.
SAS Institute.
SAS/STAT User's Guide (6.03 ed). Cary, NC: SAS Institute, 1988, p. 675-712.
33.
Schwartz, LM,
Jennings RB,
and
Reimer KA.
Premedication with the opioid analgesic butorphanol raises the threshold for ischemic preconditioning in dogs.
Basic Res Cardiol
92:
106-114,
1997[ISI][Medline].
34.
Takano, H,
Bolli R,
Black RG, Jr,
Kodani E,
Tang XL,
Yang Z,
Bhattacharya S,
and
Auchampach JA.
A(1) or A(3) adenosine receptors induce late preconditioning against infarction in conscious rabbits by different mechanisms.
Circ Res
88:
520-528,
2001
35.
Takano, H,
Manchikalapudi S,
Tang XL,
Qiu Y,
Rizvi A,
Jadoon AK,
Zhang Q,
and
Bolli R.
Nitric oxide synthase is the mediator of late preconditioning against myocardial infarction in conscious rabbits.
Circulation
98:
441-449,
1998
36.
Takano, H,
Tang XL,
and
Bolli R.
Differential role of KATP channels in late preconditioning against myocardial stunning and infarction in rabbits.
Am J Physiol Heart Circ Physiol
279:
H2350-H2359,
2000
37.
Utsumi, T,
Klostergaard J,
Akimaru K,
Edashige K,
Sato EF,
and
Utsumi K.
Modulation of TNF-alpha-priming and stimulation-dependent superoxide generation in human neutrophils by protein kinase inhibitors.
Arch Biochem Biophys
294:
271-278,
1992[ISI][Medline].
38.
Vahlhaus, C,
Schulz R,
Post H,
Rose J,
and
Heusch G.
Prevention of ischemic preconditioning only by combined inhibition of protein kinase C and protein tyrosine kinase in pigs.
J Mol Cell Cardiol
30:
197-209,
1998[ISI][Medline].
39.
Van der Geer, P,
Hunter T,
and
Lindberg RA.
Receptor protein-tyrosine kinases and their signal transduction pathways.
Annu Rev Cell Biol
10:
251-337,
1994[ISI][Medline].
40.
Vera, JC,
Reyes AM,
Velasquez FV,
Rivas CI,
Zhang RH,
Strobel P,
Slebe JC,
Nunez-Alarcon J,
and
Golde DW.
Direct inhibition of the hexose transporter GLUT1 by tyrosine kinase inhibitors.
Biochemistry
40:
777-790,
2001[Medline].
41.
Vondriska, TM,
Zhang J,
Song C,
Tang XL,
Cao X,
Baines CP,
Pass JM,
Wang S,
Bolli R,
and
Ping P.
Protein kinase C epsilon-Src modules direct signal transduction in nitric oxide-induced cardioprotection: complex formation as a means for cardioprotective signaling.
Circ Res
88:
1306-1313,
2001
42.
Wallenstein, S,
Zucker CL,
and
Fleiss JL.
Some statistical methods useful in circulation research.
Circ Res
47:
1-9,
1980
43.
Xuan, YT,
Guo Y,
Han H,
Zhu Y,
and
Bolli R.
An essential role of the JAK-STAT pathway in ischemic preconditioning.
Proc Natl Acad Sci USA
98:
9050-9055,
2001
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, Individual rabbits; 
,
means ± SE; n, number of rabbits.
0.22 (r = 0.84); and
group IV, y = 0.50x + 0.04 (r = 0.81). Analysis of covariance demonstrated that
the slope of the regression line for group II was
significantly different (P < 0.05 for each comparison)
from those for groups I and IV, indicating that
for any given risk region size, infarct size was smaller in group
II compared with groups I and IV. These data
demonstrate that late PC reduced infarct size independent of risk
region size and that this effect was abrogated by administration of
LD-A on day 2, but not by LD-A was given on day
1, or when given on day 2 without prior PC
ischemia.
