© 1999 American Heart Association, Inc.
Integrative Physiology |
Bifunctional Role of Protein Tyrosine Kinases in Late Preconditioning Against Myocardial Stunning in Conscious Rabbits
From the Experimental Research Laboratory, Division of Cardiology, University of Louisville and Jewish Hospital Heart and Lung Institute, Louisville, Ky.
Correspondence to Roberto Bolli, MD, Division of Cardiology, University of Louisville, Louisville, KY 40292. E-mail rbolli{at}louisville.edu
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Abstract |
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Abstract—Although protein tyrosine kinases (PTKs) have been implicated in late preconditioning (PC) against infarction, their role in late PC against stunning is unknown. Furthermore, it is unknown whether PTK signaling is necessary only to trigger late PC on day 1 or also to mediate it on day 2. Thus, conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3). In the control group (group I, n=7), the recovery of systolic wall thickening after the 6 occlusion/reperfusion cycles was markedly improved on days 2 and 3 compared with day 1, indicating the development of late PC against stunning. Administration of the PTK inhibitor lavendustin-A (LD-A, 1 mg/kg IV) before the first occlusion on day 1 (group II, n=7) completely prevented the late PC effect against stunning on day 2. Late PC against stunning was also abrogated when LD-A was given before the first occlusion on day 2 (group III, n=7); however, in these rabbits, the late PC effect became apparent on day 3, indicating that LD-A itself did not have any delayed deleterious actions on myocardial stunning. In group V (n=5), the sequence of 6 occlusion/reperfusion cycles resulted in a robust increase in the activity of inducible NO synthase (iNOS [assessed as Ca2+-independent L-citrulline formation]) and nitrite+nitrate (NOx) tissue levels 24 hours later (on day 2), with no concomitant change in Ca2+-dependent NO synthase (endothelial NO synthase and/or neuronal NO synthase) activity. Similar results were obtained on day 3 (group VIII, n=6), indicating sustained upregulation of iNOS. Administration of LD-A either on day 1 (group VI, n=5) or on day 2 (group VII, n=6) abrogated the increase in iNOS activity and NOx levels on day 2. LD-A had no effect on iNOS activity or NOx levels in the absence of PC (group X, n=5). This study demonstrates that in conscious rabbits, PTK activity is necessary not only to trigger late PC against stunning on day 1 but also to mediate the protection on day 2. This investigation also provides the first direct evidence that cardiac iNOS activity is upregulated during the late phase of ischemic PC in rabbits. Furthermore, the data indicate that PTK signaling is essential for the augmentation of iNOS activity and that PTKs modulate this enzyme at two distinct levels: at an early stage on day 1 and at a late stage on day 2. This bifunctional role of PTKs in late PC has broad implications for the signaling mechanisms that underlie the response of the heart to ischemic stress and, possibly, other stresses.
Key Words: myocardial stunning • protein tyrosine kinase • lavendustin A • inducible nitric oxide synthase • ischemia/reperfusion injury
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Introduction |
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Ischemic preconditioning (PC) confers myocardial protection in two temporally distinct phases: an early phase, which develops immediately and lasts for 2 to 4 hours after the ischemic stimulus, and a late phase, which begins after 12 to 24 hours and lasts for 3 to 4 days.1 2 3 Recent studies indicate that NO plays a dual role (trigger and mediator) in late PC.1 4 5 6 7 8 Specifically, the generation of NO associated with an ischemic stress initiates a cascade of events that involves the activation of several kinases and transcription factors and culminates in the upregulation of the inducible NO synthase (iNOS) gene; NO produced by iNOS then confers cardioprotection during subsequent ischemic insult(s) (reviewed in Reference 88 ). Although reactive oxygen species9 10 and protein kinase C (PKC)11 12 13 14 are known to be involved in the genesis of late PC, the exact signal transduction mechanism whereby a brief ischemic stress leads to subsequent cardioprotection remains unclear. Elucidation of the cellular basis of late PC is an essential step toward harnessing this cardioprotective phenomenon for therapeutic benefit.
Protein tyrosine kinases (PTKs), a diverse family of enzymes that transfer phosphate from ATP to tyrosine residues on specific cellular proteins, are known to mediate a wide variety of cellular responses.15 16 17 The nonreceptor PTKs are specifically designed for signal transduction from cell surface to intracellular enzymes and factors, usually by protein-protein interactions.16 Recent evidence indicates that PTKs play a role in the signaling mechanism underlying the early18 19 20 21 and the late22 phases of PC. Specifically, in isolated rat hearts, inhibition of PTKs has been found to block the PC-induced increase in the activity of phospholipase D (PLD), PKC, and mitogen-activated protein kinase–activated protein kinase 2 (MAPKAPK 2) as well as the cardioprotective effects of early PC,19 suggesting that the PTK-PLD-PKC-MAPKAPK 2 signaling pathway plays a role in the early phase of PC. Using isolated rabbit hearts, Baines et al20 have demonstrated that the protection afforded by early PC against myocardial infarction is abrogated by the PTK inhibitors genistein and lavendustin A (LD-A). In addition, genistein has been reported to block the development of late PC against infarction in open-chest rabbits.22
Although these studies18 19 20 21 22 implicate a PTK-dependent pathway in the genesis of early and late PC, a number of important issues remain to be addressed: First, while the available evidence suggests an involvement of PTKs in late PC against myocardial infarction,22 virtually nothing is known regarding the role of PTKs in the genesis of late PC against myocardial stunning. Myocardial stunning and infarction represent two very different types of injury, so that the effects of PC on one cannot be extrapolated to the other. For example, in dogs, the early phase of PC confers powerful protection against myocardial infarction23 24 but fails to protect against the stunning induced by a 10- or 15-minute coronary occlusion.25 26 27 Conversely, in conscious pigs, a sequence of ten 2-minute coronary occlusions elicits a late PC effect against stunning9 28 29 but not against infarction.30 These examples of a dissociation between the effects of PC on stunning and infarction underscore the notion that, at least under certain experimental conditions, different mechanisms may be involved in the PC protection against reversible and irreversible ischemic injury.25 Second, the precise role(s) of PTKs in late PC has not been fully elucidated. In this regard, it is important to distinguish the cellular mechanisms that initiate the development of late PC immediately after the first ischemic stress (day 1) from those that mediate cardioprotection 24 to 72 hours later (days 2 to 4). Although one previous study22 examined the effect of genistein on the development of late PC against infarction on day 1, the role of PTKs as mediators of cardioprotection on days 2 to 4 has not been explored. Thus, it is unknown whether PTK-dependent signaling is important only to trigger or also to mediate late PC. Third, virtually nothing is known regarding the cellular mechanisms by which PTKs contribute to late PC on day 1 or day 2. Although several studies have implicated iNOS as the mediator of late PC,5 7 8 direct evidence that iNOS activity is augmented in the rabbit model of late PC is still lacking, and the role of PTKs in iNOS modulation is unknown.
The present study was undertaken to address these issues. We tested the hypothesis that PTKs play a dual role in the pathophysiology of late PC against myocardial stunning, ie, 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. We further hypothesized that the mechanism by which PTKs contribute to late PC is the modulation of iNOS activity. Accordingly, the present study had three aims. First, we determined whether administration of the PTK inhibitor LD-A before the first ischemic stress (on day 1) blocks the development of late PC against myocardial stunning. Second, we investigated whether administration of LD-A before the second ischemic stress (on day 2) abrogates the cardioprotection afforded by late PC against stunning. Finally, we assessed whether LD-A (given on day 1 or day 2) interferes with the increase in iNOS activity that underlies the cardioprotection afforded by late PC. All studies were performed in conscious rabbits. The rationale for using a conscious animal model was to obviate potential problems resulting from factors associated with open-chest preparations, such as the exaggerated generation of reactive oxygen species after myocardial ischemia/reperfusion,31 which could have a major impact on the severity of myocardial stunning,31 32 and the trauma of a thoracotomy and the ensuing inflammatory reaction, which may lead to release of cytokines. LD-A was chosen because (1) this agent is more selective for PTKs than other inhibitors33 34 and (2) it has previously been shown to inhibit Src and Lck PTKs in our conscious rabbit model of late PC,35 which enabled us to assess the functional significance of Src and Lck PTK activation in this cardioprotective phenomenon.
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Materials and Methods |
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Experimental Protocol
Phase A: Studies of Myocardial Stunning
The experimental protocol consisted of 3 consecutive days (days 1, 2, and 3, respectively) of coronary artery occlusions (a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles) (Figure 1
). Rabbits
were assigned to 3 groups (Figure 1): group I (control) did not
receive any treatment; groups II and III received an
intravenous bolus of LD-A (1 mg/kg) 10 minutes before the
first coronary occlusion on day 1 or day 2, respectively.
Regional myocardial function was assessed as systolic
thickening fraction, as previously described.4 5 36 At the
conclusion of the study, the size of the occluded/reperfused
coronary vascular bed was determined by postmortem
perfusion.4 5
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Phase B: Studies of NOS Activity
Rabbits were assigned to 7 groups: group IV (sham control),
group V (PC-day 2), group VI (PC-day 2+LD-A on day 1), group VII
(PC-day 2+LD-A on day 2), group VIII (PC-day 3), group IX (PC-day
3+LD-A on day 2), and group X (LD-A without PC) (Figure 1
).
Rabbits in group IV did not receive coronary
occlusion/reperfusion and were euthanized a minimum of 10 days after
surgery. Rabbits in groups V, VI, and VII underwent a sequence of six
4-minute occlusion/4-minute reperfusion cycles as in phase A; in groups
VI and VII, LD-A (1 mg/kg IV) was administered 10 minutes before the
first coronary occlusion on day 1 and day 2, respectively.
Rabbits in groups V and VI were euthanized 24 hours later. Rabbits in
group VII were euthanized 25 minutes after the administration of LD-A
(time interval corresponding to the interval elapsed between the
administration of LD-A and the mid-part of the 6 occlusion/reperfusion
cycles in group III). Rabbits in groups VIII and IX underwent a
sequence of six 4-minute occlusion/4-minute reperfusion cycles on days
1 and 2 and were euthanized 24 hours later; in group IX, LD-A was given
on day 2 as in group III (1 mg/kg IV 10 minutes before the first
coronary occlusion). Rabbits in group X received the same dose
of LD-A (1 mg/kg IV) and were euthanized 25 minutes later (similar to
group VII). In all 7 groups, the heart was excised, and myocardial
samples (
0.5 g) were rapidly removed from the
ischemic/reperfused and the nonischemic region and
frozen in liquid nitrogen.
Measurement of NOS Activity
Tissue samples were homogenized in the appropriate
buffer, and cytosolic and membrane fractions were isolated.
Ca2+-dependent (cNOS) and
Ca2+-independent (iNOS) NOS activities were
determined by measuring the conversion of
[14C]L-arginine to
[14C]L-citrulline using a
modification of the procedure of Bredt and Snyder.37
Measurement of Nitrite and Nitrate
Tissue nitrite was assayed by using the Griess reaction as
modified by Gilliam et al.38 Tissue nitrate was determined
after conversion of nitrate to nitrite with Aspergillus
nitrate reductase.38 All assays were performed in
duplicate.
Statistical Analysis
Data are reported as mean±SEM. One-way or two-way
repeated-measures ANOVA followed by Student’s t tests for
paired or unpaired data with the Bonferroni correction was used, as
appropriate.39 40
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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A total of 63 conscious rabbits were used in the present study (2 for the pilot studies, 23 for phase A, and 38 for phase B).
Pilot Studies
Pilot studies were conducted in 2 rabbits to identify a dose of
LD-A that has no effect on heart rate, arterial blood
pressure, and systolic wall thickening (WTh). The concern was
that hemodynamic perturbations caused by LD-A (eg, a
fall in blood pressure or an increase in heart rate) could
nonspecifically induce a late PC effect unrelated to the
ischemic stimulus. Arterial pressure was measured
by cannulating the dorsal ear artery with a 22-gauge angiocatheter
under local anesthesia (benzocaine), as previously
described.4 5 12 The IC50 of LD-A
for receptor PTKs (eg, epidermal growth factor receptor) is 0.011
µmol/L, while the IC50 for nonreceptor PTKs
(pp60c-src) is 0.5
µmol/L.33 34 We initially tested a dose of 0.45 mg/kg
IV, which was calculated to produce plasma concentrations 5 times
the IC50 of LD-A against nonreceptor PTKs. This
dose did not have any hemodynamic effect when
administered on day 1 (before the PC occlusion/reperfusion protocol) or
(in another rabbit) on day 2, before the second ischemic
challenge; however, this dose failed to block late PC against stunning.
Therefore, we increased the dose to 1 mg/kg (10 times the
IC50 for nonreceptor PTKs); this dose did not
have any significant effect on blood pressure or heart rate but did
block late PC when given on day 1 or day 2. Doses higher than 1 mg/kg
would have been prohibitively expensive. Consequently, a dose of 1
mg/kg was chosen for the present experiments.
Phase A: Studies of Myocardial Stunning
Exclusions and Postmortem Analysis
Of the 23 rabbits instrumented for phase A, 7 were assigned to
group I (control group), 9 to group II (LD-A on day 1), and 7 to group
III (LD-A on day 2). All animals assigned to the control group
completed the protocol on days 1, 2, and 3. Of the 9 rabbits assigned
to group II, 2 were excluded because of persistent dyskinesis after the
sixth reperfusion on day 1
(triphenyltetrazolium chloride staining
demonstrated myocardial infarction that was most likely due to
malfunction of the occluder), and 1 died on day 3 because of
ventricular fibrillation during the fourth occlusion.
Therefore, 6 rabbits in group II completed days 1, 2, and 3, whereas 1
rabbit completed only days 1 and 2. Of the 7 rabbits assigned to group
III, 1 died of ventricular fibrillation on day 3 during the
fifth occlusion. Therefore, 6 rabbits in group III completed days 1, 2,
and 3.
Postmortem analysis showed that the size of the occluded/reperfused vascular bed was similar in the 3 groups: 0.79±0.13 g (17.5±2.6% of left ventricular [LV] weight) in group I, 0.83±0.09 g (16.1±1.5% of LV weight) in group II, and 0.84±0.07 g (15.8±1.6% of LV weight) in group III. Tissue staining with triphenyltetrazolium chloride confirmed the absence of infarction in all animals included in the final analysis. In all rabbits, the ultrasonic crystal was found to be at least 3 mm from the boundaries of the ischemic/reperfused region.
Regional Myocardial Function
As shown in the Table, there were no
appreciable differences in heart rate among the 3 groups, either during
the sequence of coronary occlusion/reperfusion cycles or during
the 5-hour reperfusion period. These results are in agreement with our
pilot studies and confirm that the dose of LD-A selected in this study
has no effect on hemodynamic variables in conscious
rabbits. Baseline systolic thickening fraction in the region to
be rendered ischemic averaged 35.0±4.9%, 33.8±4.0%, and
34.4±4.2% on days 1, 2, and 3, respectively, in group I; 41.0±3.2%,
40.2±3.8%, and 39.7±4.1% in group II; and 38.5±3.8%, 37.8±3.4%,
and 37.7±4.3% in group III (Figures 2, 3, and 4).
There were no significant differences among the 3 groups on the same
day or among different days within the same group. In group II,
thickening fraction on day 1 was 41.0±4.2% at baseline and
39.4±3.4% after administration of LD-A (preocclusion)
(P=NS, Figure 3), indicating that this agent had no
significant effect on regional myocardial function. This conclusion is
further corroborated by the results in group III, in which thickening
fraction on day 2 was 37.8±3.4% at baseline and 37.7±3.6% after
administration of LD-A (P=NS, Figure 4).
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Group I (Control)
On day 1, thickening fraction remained significantly
(P<0.05) depressed for 3 hours after the sixth reperfusion
and recovered by 5 hours (Figure 2), indicating that the
sequence of six 4-minute occlusion/4-minute reperfusion cycles resulted
in severe myocardial stunning that lasted, on average, 4 hours. On days
2 and 3, however, the recovery of WTh was markedly improved after the 6
occlusion/reperfusion cycles compared with day 1 (Figure 2). The
total deficit of WTh after the sixth reperfusion was 52% and 53% less
on days 2 and 3, respectively, compared with day 1 (P<0.01)
(Figure 5). Thus, as
expected,4 5 12 41 myocardial stunning was attenuated
markedly, and to a similar extent, on days 2 and 3 compared with day
1.
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Group II (LD-A on Day 1)
On day 1, the recovery of WTh (Figure 3) and the total
deficit of WTh (Figure 5) were similar to values observed in the
control group, indicating that LD-A had no appreciable effect on the
severity of myocardial stunning in nonpreconditioned
myocardium. On day 2, however, the recovery of WTh during
the 5-hour period after the sixth reperfusion was not improved compared
with day 1 (Figure 3), and the total deficit of WTh on day 2 was
not significantly different from that observed on day 1 (Figure 5). The total deficit of WTh on day 2 was 90% greater than the
corresponding value in control rabbits (P<0.01) and similar
to that observed in control rabbits on day 1 (Figure 5). On day
3, the recovery of WTh in LD-A–treated rabbits was markedly improved
compared with day 2 (Figure 3) and was similar to that noted on
day 2 in the control group (Figure 2). The total deficit of WTh
was 66% less than that noted on day 2 in the same animals
(P<0.01) and was comparable to that noted on day 2 in
control rabbits (Figure 5). Thus, the sequence of 6
coronary occlusions and reperfusions performed on day 1 after
the administration of LD-A failed to induce late PC against stunning on
day 2, but the same sequence performed on day 2 did precondition
against stunning on day 3.
Group III (LD-A on Day 2)
On day 1, the recovery of regional function was similar to that
observed in the control group (Figures 4 and 5). On day
2, however, both the recovery of WTh (Figure 4) and the total
deficit of WTh (Figure 5) were similar to values observed on day
1. The total deficit of WTh on day 2 was 86% greater than the
corresponding value in control rabbits (P<0.01) and was
similar to that observed in control rabbits on day 1 (Figure 5).
Thus, administration of LD-A on day 2 completely abrogated the
protective effects of late PC. On day 3, the recovery of WTh was
markedly improved compared with day 2 (Figure 4) and was similar
to that observed on day 3 in the control group (Figure 2). The
total deficit of WTh was 59% less than that noted on day 2 in the same
animals (P<0.01) and was comparable to that noted on day 3
in control rabbits (Figure 5). Thus, administration of LD-A
before the sequence of 6 coronary occlusions and reperfusions
on day 2 completely abolished the late PC effect against stunning that
was induced by the ischemic stimulus on day 1. The significant
improvement in the recovery of WTh noted on day 3 in these rabbits
indicates that LD-A in itself did not have any delayed deleterious
effects on myocardial stunning.
Phase B: Studies of NOS Activity
Having observed in phase A that late PC is blocked by the
administration of LD-A on day 1 as well as on day 2, in phase B we
investigated whether PTKs participate in late PC by modulating iNOS
activity.
Exclusions
Of the 38 rabbits instrumented for phase B, 5 were assigned to
groups IV, V, VI, IX, and X, 6 to group VIII, and 7 to group VII. One
rabbit in group VII was excluded from analysis because of
probable infarction.
Effect of Ischemic PC on NOS Activity and Myocardial
Nitrite+Nitrate (NOx) Levels
As detailed in Materials and Methods, NOS activity was
measured as
NG-nitro-L-arginine
methyl ester (L-NAME)–inhibitable L-citrulline
production. In group V (ischemic PC-day 2), the
sequence of 6 cycles of 4-minute occlusion/4-minute reperfusion on day
1 resulted in a robust induction of iNOS activity 24 hours later (on
day 2). In these rabbits, total (cytosolic+membranous fractions)
Ca2+-independent NOS activity (iNOS activity) in
the ischemic/reperfused region was 167% higher than the
activity in the nonischemic region (P<0.05) and
119% higher than the activity in the anterior LV wall in sham control
rabbits (group IV) (P<0.05) (Figure 6). The increase in iNOS activity was
more pronounced in the cytosolic fraction (+233%, P<0.05)
than in the membranous fraction (+100%, P<0.05). No
significant changes in iNOS activity were observed in the
nonischemic region (Figure 6). In contrast to iNOS
activity, Ca2+-dependent NOS
(endothelial NOS and/or neuronal NOS) activity in the
ischemic/reperfused region did not change appreciably 24 hours
after ischemic PC (on day 2), either in the total
homogenate (cytosolic+membranous fractions) (Figure 7) or in the individual (cytosolic and
membranous) tissue fractions (data not shown). The changes in iNOS
activity were associated with directionally concordant changes in the
tissue levels of nitrite+nitrate (NOx), the
stable oxidation products of NO: as illustrated in Figure 8, in group V total (cytosolic+membranous
fractions) myocardial NOx levels in the
ischemic/reperfused region were significantly increased 24
hours after ischemic PC (day 2) (+62% versus the
nonischemic region [P<0.05] and +43% versus the
anterior LV wall in group IV [sham controls]
[P<0.05]).
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In group VIII (ischemic PC-day 3), rabbits were subjected to a
sequence of six 4-minute occlusion/reperfusion cycles on day 1 and to
another sequence on day 2; 24 hours later (on day 3), the increases in
iNOS activity (Figure 6) and NOx levels
(Figure 8) were similar to those measured on day 2 in group V
(ischemic PC-day 2). No change in cNOS activity was noted on
day 3 (Figure 7). Thus, myocardial iNOS activity and
NOx levels were augmented both on day 2 and on
day 3, indicating sustained upregulation of iNOS by late PC.
Effect of LD-A on NOS Activity and Myocardial
NOx
In the absence of ischemic PC (group X), LD-A did not have
any appreciable effect on iNOS activity (Figure 6), cNOS
activity (Figure 7), or NOx levels (Figure 8). When LD-A was administered before the PC ischemia on
day 1 (group VI), iNOS activity and NOx levels in
the ischemic/reperfused region 24 hours later (day 2) were not
significantly different from those measured in the nonischemic
region in the same group and in the anterior LV wall of sham control
rabbits (group IV) (Figures 6 and 8). Both iNOS activity
and NOx levels were significantly less in
preconditioned rabbits treated with LD-A on day 1 (group VI) compared
with untreated preconditioned rabbits (group V) (Figures 6 and 8). Thus, pretreatment with LD-A completely abrogated the
ischemic PC–induced increase in iNOS activity and
NOx levels observed 24 hours later (day 2) in
group V.
When LD-A was administered on day 2 (group VII), iNOS activity and
NOx levels in the ischemic/reperfused
region 25 minutes after LD-A did not differ significantly from those
measured in the anterior LV wall in sham control rabbits (group IV) and
were significantly less (P<0.05) than those measured in
rabbits subjected to ischemic PC (group V) (Figures 6 and 8). Thus, administration of LD-A 24 hours after the initial
PC ischemia (day 2) markedly suppressed the ischemic
PC–induced increase in iNOS activity and abrogated the
ischemic PC–induced increase in myocardial
NOx levels on day 2. In contrast, administration
of LD-A on day 2 had no significant effect on the ischemic
PC–induced increase in iNOS activity and NOx
levels observed 24 hours later (on day 3) (group IX).
Administration of LD-A either on day 1 or on day 2 had no discernible
effect on cNOS activity (Figure 7).
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Discussion |
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Although previous studies have identified the initial components of the signal transduction pathway that underlies late PC, including NO,4 5 6 7 reactive oxygen species,9 10 and PKC,11 12 13 14 the mechanisms downstream from these molecules are poorly understood. Recent studies have shown that ischemic PC activates Src and Lck PTKs,23 but the functional significance of this observation remains unknown. In particular, no information is available regarding whether PTKs are causally involved in late PC against myocardial stunning and, if so, whether they participate in the development of this adaptive phenomenon on day 1 and/or in the manifestation of its cardioprotective effects on day 2. Furthermore, no information is available regarding the mechanism whereby PTKs contribute to late PC.
Salient Findings
The present investigation provides significant new
insights regarding these issues. Using conscious rabbits, we found that
administration of LD-A before the ischemic PC stimulus (on day
1) completely blocked the development of protection against myocardial
stunning 24 hours later, indicating that PTK activity is necessary to
trigger this mechanism. The protective effects of late PC against
stunning were also abrogated when LD-A was administered before
ischemia/reperfusion on day 2, indicating that PTK activity is
also necessary to mediate this phenomenon. To our knowledge, this is
the first indication that PTKs are involved in the manifestation of
protection during the late phase of ischemic PC (on day 2).
Finally, administration of LD-A either on day 1 or on day 2 abrogated
the increase in myocardial iNOS activity and NOx
levels on day 2, indicating that the upregulation of iNOS (which
mediates the cardioprotective effects of late PC1 5 7 8 )
occurs via at least two PTK-dependent pathways, one that is operative
on day 1 and one that is active on day 2.
Previous studies have implicated PTKs in early18 19 20 21 and late22 PC against myocardial infarction. To our knowledge, this is the first study to demonstrate that PTKs play an obligatory role in the development of late PC against myocardial stunning. This is also the first study (1) to identify two distinct functions for PTKs in late PC against stunning (ie, PTKs are required both to trigger this phenomenon on day 1 and to mediate it on day 2), (2) to directly demonstrate that iNOS activity is upregulated during the late phase of ischemic PC in rabbits, (3) to indicate that this upregulation of iNOS activity requires PTK activity, and (4) to examine the effect of PTK inhibitors on ischemic PC in conscious animals. The finding that cardiac iNOS upregulation is PTK dependent reveals a new signaling mechanism and a new function for PTKs, which has significant implications for many pathophysiological processes besides ischemic PC.
Rationale for Selecting LD-A
There were several reasons for selecting LD-A instead of
genistein as a PTK inhibitor. First, the selectivity of
LD-A for PTKs versus other kinases is superior to that of genistein.
The IC50 ratio of genistein for nonreceptor PTKs
versus protein kinase A/PKC has been found to be 1:10 to
1:14,34 42 whereas LD-A is a highly selective
inhibitor of PTKs, with an IC50 of
0.011, 0.5, and >100 µmol/L for receptor PTKs, nonreceptor
PTKs, and serine-threonine kinases (including PKC, protein kinase A,
and calmodulin-dependent kinases),
respectively.33 34 Thus, the IC50
ratio for nonreceptor PTKs versus PKC is <1:200 for
LD-A34 compared with <1:10 for genistein. The dose of
LD-A selected for the present study (1 mg/kg) was calculated to
result in peak plasma concentrations of 4.5 µmol/L, 10 times
higher than the IC50 for Src PTKs (0.5
µmol/L).33 34 Because in intact organisms LD-A is
redistributed after intravenous injection, the plasma and
tissue levels present during the 6 occlusion/reperfusion cycles 10
to 54 minutes after the intravenous bolus of LD-A should
have been considerably lower, making it very unlikely that the observed
inhibition of late PC could be ascribed to inhibition of PKC or other
serine-threonine kinases.
The second advantage of LD-A over genistein is that it is much more selective for the Src family of PTKs. Genistein and other flavonoids are general inhibitors of PTKs with little specificity for individual enzymes.43 In contrast, LD-A preferentially inhibits two families of PTKs, epidermal growth factor receptor kinases and Src PTKs.33 34 In view of the fact that previous studies in our conscious rabbit model have shown that ischemic PC activates Src PTKs but has no effect on epidermal growth factor receptor PTKs,35 LD-A would appear to be a useful tool for interrogating the Src family of PTKs. The third reason for selecting LD-A was that this agent (at the same dose used in the present study) has been documented to block Src PTK activation during ischemic PC.35 In view of these facts, the actions of LD-A documented in the present study suggest a critical role of the Src family of PTKs both in the development of late PC on day 1 and in the manifestation of its cardioprotective effects on day 2.
Role of PTKs in the Development of Late PC (Day 1)
Recent studies in conscious rabbits have identified a
pivotal role of the transcription regulatory protein nuclear factor
(NF)-
B in the development of late PC on day 1.41 These
studies have also found that the activation of NF-B by
ischemic PC was prevented by pretreatment with LD-A (given at
the same dose that was used in the present investigation),
indicating that the recruitment of this transcription factor requires
PTK signaling.41 However, the mechanism whereby
PTK-dependent recruitment of NF-B produces delayed cardioprotection
remained unknown. The present findings that pretreatment with LD-A
on day 1 abrogated the increase in iNOS activity and tissue
NOx levels 24 hours later (Figures 6 and 8) indicate that PTK signaling on day 1 is required for the
upregulation of iNOS on day 2. NF-B is known to be a central
mechanism controlling iNOS induction,44 45 and mounting
evidence indicates that the late phase of PC is mediated by iNOS
activity.5 7 8 Thus, based on the fact that the
mobilization of PTKs shortly after the initial PC stimulus (on day 1)
is essential not only for the activation of NF-B, as shown
previously,41 but also for the increase in iNOS activity
and for the acquisition of cardioprotection 24 hours later, as shown in
the present study (Figures 5, 6, and 8), we
propose that PTKs participate in the genesis of late PC by promoting
iNOS transcription via an NF-B–dependent pathway.
This paradigm is consistent with emerging evidence in
noncardiac cells, in which tyrosine phosphorylation has
been found to be necessary for NF-B mobilization. For example, the
PTK inhibitors herbimycin A and genistein can block
hypoxia-induced phosphorylation of
inhibitory B
(IB) on tyrosine residues and the
consequent activation of NF-B in Jurkat T cells.46
Recently, it has been shown that tyrosine
phosphorylation of IB induced by the phosphatase
inhibitor pervanadate promotes NF-B DNA-binding activity
without degradation of IB.47 In that study, a PTK of
the Src family (Lck) was proven essential for pervanadate-induced
IB tyrosine phosphorylation and NF-B
activation.47 Interestingly, Lck is activated by
ischemic PC in conscious rabbits, and the activation is blocked
by LD-A, given at the same dose as in the present
study,35 raising the possibility that this specific kinase
may be involved in the ischemic PC–induced recruitment of
NF-B.
In the present study, iNOS activity was augmented not only on
day 2 (group V) but also on day 3 (group VIII), demonstrating sustained
upregulation of this enzyme during late PC. The mechanism for the
increased iNOS activity of day 3 appears to be different from that on
day 2, in view of the fact that the administration of LD-A on day 2 had
no significant effect on myocardial stunning (Figures 4 and 5), iNOS activity (Figure 6), and
NOx levels (Figure 8) 24 hours later (on
day 3) (group III and IX). Thus, although the protection against
stunning and the concomitant increase in iNOS activity observed on day
2 require PTK signaling on day 1 (groups II and VI), it appears that
neither the protection against stunning nor the concomitant
upregulation of iNOS activity observed on day 3 is dependent on PTK
signaling on day 2 (groups III and IX). These results are congruent
with the finding that, in conscious rabbits, a single sequence of six
4-minute occlusion/reperfusion cycles elicits a state of PC against
stunning that lasts for 72 hours even when no additional
ischemic PC stimuli are applied.48 Together with
this previous study,48 the present results suggest
that both the preconditioned state and the rise in iNOS activity seen
on day 3 are induced by the initial PC stimulus on day 1 and,
therefore, do not require any signaling events on day 2.
Role of PTKs in the Mediation of Late PC (Day 2)
A novel finding of the present study was that LD-A abolished
the cardioprotective effects of late PC when given on day 2, after the
preconditioned state had developed, revealing a role of PTK signaling
24 hours after the initial ischemic stress. To our knowledge,
this is the first indication that the activity of a cellular kinase is
involved in the mediation (as opposed to the triggering) of delayed
protection after a PC stimulus. This finding impels a reassessment of
current paradigms, because it implies that the augmented iNOS activity
that underlies the late phase of PC does not result exclusively from
increased iNOS transcription49 but must also involve other
mechanisms. Specifically, the fact that both the increase in myocardial
iNOS activity (Figure 6) and the concomitant increase in
NOx levels (Figure 8) were abrogated by
the administration of LD-A on day 2 suggests that in addition to the
synthesis of new iNOS proteins,50 posttranslational
modulation of iNOS proteins via tyrosine
phosphorylation is also critical in activating this
enzyme and conferring enhanced tolerance to myocardial
ischemia/reperfusion injury. Thus, a complex scenario emerges
from the present observations, in which PTK signaling elicited by
brief ischemia upregulates iNOS both via increased
transcription (day 1) and via posttranslational modulation (day 2).
The concept that tyrosine phosphorylation is required for stress-induced iNOS proteins to be protective reveals a new function of PTKs in cardiac pathophysiology. This concept is congruent with a previous study51 in which tyrosine phosphorylation of iNOS in murine macrophages was found to be associated with increased iNOS activity. Further studies will be needed to elucidate the mechanism whereby PTK activity is enhanced on day 2 and to identify the specific kinase(s) involved.
Conclusions
The present study provides new insights into the role of
PTKs in cardioprotection. The results of phase A demonstrate that, in
conscious rabbits, PTKs perform two distinct functions within the
signal transduction pathway that underlies late PC against myocardial
stunning: on day 1, they are essential for the development of the late
PC effect, whereas on day 2, they are essential for its manifestation.
The results of phase B are the first direct evidence that iNOS activity
is enhanced during late PC in rabbits. Furthermore, phase B provides a
mechanistic basis for the results of phase A, in that it indicates that
PTKs are involved in the modulation of cardioprotective iNOS activity
at two distinct levels: at an early stage on day 1 and at a late stage
on day 2. This heretofore unrecognized bifunctional role of PTKs
suggests a new pathophysiological paradigm for the
late phase of ischemic PC and has broad implications for our
understanding of the signaling mechanisms that underlie the response of
the heart to ischemic stress and, possibly, other stresses. The
present findings support the concept that tyrosine
phosphorylation serves as a critical regulatory
mechanism for stress-activated gene upregulation in the
heart.
|
Acknowledgments |
|---|
This study was supported in part by National Institutes of Health grants R01 HL-43151 and HL-55757 (Dr Bolli), by Kentucky American Heart Association Fellowships KY-96-F-24 and 9804556 (Dr Dawn) and 9804558 (Dr Takano), and by the Jewish Hospital Research Foundation, Louisville, Ky. We gratefully acknowledge Gregg Shirk and Wen-Jian Wu for expert technical assistance and Trudy Keith for expert secretarial assistance.
|
Footnotes |
|---|
This manuscript was sent to Stephen F. Vatner, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received June 22, 1999; accepted September 27, 1999.
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References |
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