© 1999 American Heart Association, Inc.
Original Contributions |
Emigrated Neutrophils Regulate Ventricular Contractility via 
4 Integrin
From the Department of Physiology and Biophysics and Immunology Research Group, University of Calgary, Calgary, Alberta, Canada.
Correspondence to P. Kubes, Immunology Research Group, Health Sciences Centre, University of Calgary, 3330 Hospital Dr NW, Calgary, Alberta T2N 4N1, Canada. E-mail pkubes{at}ucalgary.ca
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Abstract |
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Abstract—We have previously shown that CD18 and
4 integrin were important in the
adherence of emigrated neutrophils to cardiac myocytes. Whether either
of these molecules is important in myocyte dysfunction is unclear. In
this study, we measured contractility as an index of
myocyte function. Control contractility was compared
with shortening response in myocytes exposed to neutrophils in the
presence and absence of anti-CD18 or anti-4 antibodies.
Control unloaded cell shortening, expressed as a percentage of resting
cell length, measured 10.06±1.16% (n=10) at 5 minutes. Circulating
neutrophils caused a 35% reduction in cell shortening, an event
prevented by anti-CD18, but not by anti-4 antibody. When
emigrated neutrophils were added to the myocytes, a profound reduction
(50%) in unloaded cell shortening was noted. A significant increase in
CD18 and 4 integrin was found on emigrated neutrophils.
Addition of anti-CD18 antibody did not protect the myocyte from the
emigrated neutrophils, whereas the addition of an anti-4
antibody significantly reduced neutrophil-induced cell shortening,
despite some neutrophils still adhering to the myocytes. Furthermore,
emigrated neutrophils were able to cause myocytes to go into
contracture within 5 minutes in the presence of neutrophils with or
without anti-CD18 antibody. In addition to the impairment in unloaded
cell shortening, at later times (10 minutes), neutrophils also caused a
40% reduction in the rate of contraction and relaxation. The addition
of either anti-CD18 or anti-4 antibody protected the
myocytes from these changes. The data suggest that immunosuppression of
CD18 on emigrated neutrophils was only partially effective in reducing
myocyte dysfunction. In contrast, immunosuppression of the
4 integrin alone was sufficient to dramatically reduce
all parameters of cell dysfunction measured in this
study.
Key Words: myocyte • emigrated neutrophil • ischemia/reperfusion • contractility • 4 integrin
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils have been implicated as having a direct role in myocardial injury during inflammation,1 2 3 4 given that depletion of neutrophils from the circulation has been found to reduce myocardial injury after ischemia-reperfusion.5 6 7 It is thought that after neutrophils infiltrate myocardium,8 9 10 11 they release cytotoxic factors such as oxygen free radicals, proteases, and arachidonic acid metabolites.3 8 12 Targeting these molecules also reduced the extent of myocardial injury after ischemia-reperfusion.13 14 15 16 In earlier studies, a very important observation was that firm adhesion between myocytes and neutrophils was required for both the release of these toxic mediators17 18 and the subsequent injury.17 19 Detailed reports have proposed that (1) the engagement of CD18 (the ß2 integrin responsible for firm adhesion) was essential for neutrophils to release cytotoxic molecules2 18 20 21 and (2) the tight seal between the neutrophil and myocyte may exclude plasma, which contains important antioxidants and antiproteases.17 22 When neutrophil adhesion is disrupted with anti-CD18 or anti–intercellular adhesion molecule-1, plasma-derived antioxidants can prevent myocardial injury, highlighting the absolute requirement for neutrophil adhesion in this pathology.17
Although these seminal studies have convincingly demonstrated the
essential role for adhesion between circulating neutrophils and cardiac
myocytes, the chosen experimental conditions differed from the
physiological situation, inasmuch as neutrophils
must first emigrate out of the vasculature before they interact with
cardiac myocytes. The emigration process is not trivial; emigrated
neutrophils have been shown to be far more responsive to inflammatory
mediators23 24 and to express novel adhesion molecules,
including 4 integrin.25
Indeed, the 4 integrin has been shown to
contribute significantly to emigrated neutrophil-myocyte interactions.
After emigration, targeting only CD18 with an anti-CD18 antibody (Ab)
no longer inhibited adhesion.26 Rather, both anti-CD18 and
anti-4 Abs were required to prevent emigrated
neutrophil-myocyte interactions. This observation has raised many new
questions about the importance of CD18, as well as
4 integrin, as mediators of emigrated
neutrophil-dependent myocyte injury. For example, is CD18 still
required for neutrophil-induced myocyte injury if the neutrophils have
emigrated and are now expressing 4 integrin?
Does 4 integrin play a role in the injury
induced by emigrated neutrophils, or are both molecules essential for
injury to ensue?
In this study we, for the first time, examined in a systematic fashion
the role of CD18 and 4 integrin on emigrated
neutrophils as essential adhesive molecules required for myocyte
injury. Surprisingly, we demonstrate that immunosuppression of CD18 was
only partially effective in reducing myocyte dysfunction and death
after exposure to emigrated neutrophils. Immunosuppression of the
4 integrin, however, dramatically reduced cell
dysfunction and delayed myocyte death, despite the adherence of some
neutrophils in the presence of anti-4 Ab.
These data suggest that 4, and not CD18, is
the predominate molecule responsible for regulation of
adhesion-dependent cellular injury after neutrophil emigration.
Moreover, the results imply that circulating and emigrated neutrophils
use different adhesive pathways leading to cytotoxicity. Accordingly,
alternate adhesive therapeutic interventions need to be considered.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All experimental protocols were reviewed and approved by the University of Calgary Animal Resource Center.
Isolation of Ventricular Myocytes
Ventricular myocytes were isolated as previously
described for rat ventricular myocytes26 with
minor modifications for murine cells. Briefly, 6-week-old male C57BL/6
mice were anesthetized with methoxyfluorane (Metafane; Janssen
Pharmaceutica). After exsanguination, hearts were removed and placed
into Tyrode buffer (in mmol/L, NaCl 140, KCl 5.4,
Na2HPO4 1, HEPES 5, glucose
10, and MgCl2 1, pH adjusted to 7.4 with NaOH)
containing 1 mmol/L CaCl2 at 4°C. Hearts
were then cannulated via the aorta (within 3 minutes) for retrograde
perfusion of the coronary arteries. Initially, the hearts were
perfused with Tyrode buffer containing 1 mmol/L
CaCl2 at 2 mL/min for 5 minutes at 37°C and
then with Tyrode buffer containing no CaCl2 at 2
mL/min for 5 minutes. Perfusion was then switched to Tyrode buffer
containing 40 µmol/L CaCl2, 20 µg/mL
collagenase (Yakult Pharmaceuticals Co), and 4 µg/mL
protease (Sigma), and perfusion continued at 2 mL/min for 8 minutes.
Digested hearts were then removed from the perfusion system, and
ventricles were minced in Tyrode buffer containing 1 mmol/L
CaCl2, 500 µg/mL collagenase, 100
µg/mL protease, and 2.5% BSA (Sigma). Ventricular tissue
segments were then put into a shaking water bath for 10 to 20 minutes
at 37°C to complete the dispersion and obtain a suspension of
individual myocytes. Myocytes were then placed in a KB-type solution
(in mmol/L, potassium glutamate 100, potassium aspartate 10, KCl
25, KH2PO4 10,
MgSO4 2, taurine 20, creatine 5, EGTA 0.5,
glucose 20, and HEPES 5, and BSA 1%, pH adjusted to 7.2 with KOH) at
4°C and used within 5 hours. All chemicals were from BDH Inc except
HEPES (Sigma), unless otherwise stated.
Isolation of Emigrated Neutrophils
Six-week-old male C57BL/6 mice were injected
intraperitoneally with 1% oyster glycogen (Sigma)
in saline.27 After 4 hours, mice were euthanized, and a
peritoneal lavage was performed with 3 mL of saline. Lavage fluid was
placed on ice for 5 minutes and then centrifuged at 1000 rpm at
4°C for 6 minutes. Pellets were then resuspended in Tyrode buffer
with 1 mmol/L CaCl2 at 4°C. This approach
yielded a 99% pure population of emigrated neutrophils as
analyzed with Wright-Giemsa staining. In all experiments,
neutrophils were kept on ice and used within 2 hours of isolation.
Unloaded Cell Shortening Experiments
Isolated ventricular myocytes were allowed to adhere
to a glass microscope stage for 5 minutes at room temperature. Myocytes
were then superfused at 1 mL/min with normal Tyrode buffer containing
1 mmol/L CaCl2. Cells were field stimulated
at 1 Hz using a just-threshold voltage level (Isolator II, Axon
Instruments) to minimize production of free radicals due to
hydrolysis. Unloaded cell shortening was recorded using an
edge-detection device (Solamere Technology Group), and the data were
acquired digitally at a 10-kHz sampling rate using customized software
(Cellsoft version 2.0, D. Bergman, University of Calgary,
Alberta, Canada). The number of neutrophils adherent per myocyte and
the time of onset of dysrhythmia were recorded for each myocyte.
For all experiments, cells were allowed to equilibrate while being
electrically stimulated continuously for 15 minutes. To ensure that
myocytes exhibited normal contractile behavior and inotropic capacity
before neutrophil treatment, the ß-adrenergic agonist isoproterenol
(Sigma, 0.1 µmol/L) was added, and the resulting positive
inotropic response to electrical stimulation was monitored. Myocytes
exhibiting baseline shortening <5% of resting length or those failing
to respond to isoproterenol were excluded from the study. A positive
response to isoproterenol included a 2-fold increase in extent of cell
shortening, rate of contraction, and rate of relaxation at
baseline.
After isoproterenol was washed out (10 minutes), baseline measurements
were taken, and then 1x106 neutrophils,
prestimulated with 1% zymosan-activated plasma (ZAP), were
added to the superfusate. Myocyte contractility
was then recorded continuously for 10 minutes. Isoproterenol was
added again to the superfusate to reassess myocyte
contractility. In all experiments, myocyte
contractility was recorded to the completion of the
protocol unless cell death occurred. Neutrophil treatment was carried
out in the following 4 groups: (1) control (no neutrophils), (2)
polymorphonuclear leukocytes (PMNs; neutrophils only), (3)
anti-CD18 (neutrophils+anti-CD18 Ab 2E6, 8 µg/mL) (Endogen), and (4)
anti-4
(neutrophils+anti-4 Ab R1-2, 10 µg/mL)
(Pharmingen). As anti-4 Ab was sufficient to
inhibit all myocyte dysfunction, there was no need to administer both
Abs simultaneously.
In additional experiments, circulating leukocytes were isolated from
whole blood by lysis of the red blood cells. These leukocytes were
initially exposed to myocytes, and histological
assessment revealed that all of the adherent cells were indeed
neutrophils. Neutrophil adhesion to myocytes was then documented after
the addition of ZAP in the presence of anti-CD18 or
anti-4 Abs. Additional experiments examined
myocyte cell shortening in the presence of circulating leukocytes as
described for emigrated neutrophils.
Flow cytometry was used to measure the expression of CD11b, CD18, and
4 integrin on circulating and emigrated
neutrophils. Circulating or emigrated murine neutrophils
(1x106 per tube) were stimulated with ZAP (10
minutes at room temperature) and then washed. Red blood cells were
lysed, and neutrophils were fixed in 1% formalin (15 minutes at room
temp) and then washed. Primary Abs directed against their respective
adhesion molecules (CD11b, MK/170, 0.25 µg per tube; CD18, 2E6, 0.8
µg per tube; and 4, R1-2, 1 µg per tube)
were added. After 30 minutes at room temperature, cells were washed and
labeled with FITC-conjugated goat anti-rat IgG (Cedar Lanes
Laboratories Ltd) for CD11b and 4, and
FITC-conjugated goat anti-hamster IgG (Caltag Laboratories) for CD18.
After 30 minutes at room temperature, cells were washed and
fluorescence was measured on a FACScan flow cytometer (Becton
Dickinson Immunocytochemistry Systems).
Statistical Analysis
All data are expressed as the arithmetic mean±SEM. Data were
compared between treatment groups using an ANOVA of raw data with the
Dunnett method for multiple comparisons to the PMN-only group and the
Student t test within 2 groups. Values of P<0.05
are considered statistically significant.
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Results |
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Figure 1
demonstrates a
representative pattern of unloaded cell shortening
observed during the entire 10-minute protocol in myocytes that were not
exposed to neutrophils. These cells were electrically stimulated at 1
Hz, and as expected, the unloaded cell shortening at the beginning and
end of each experimental protocol remained unchanged. When the myocyte
was exposed to isoproterenol, it showed the following characteristic
positive inotropic responses to ß-adrenergic stimulation: (1) marked
increase in the extent of cell shortening, (2) faster rate of
contraction, and (3) increased rate of relaxation. These responses were
the same at the beginning and end of each experiment. In the next
series of experiments, the cells were again first exposed to
isoproterenol, and then emigrated neutrophils were added and allowed to
adhere to the myocytes. Figure 2
demonstrates a representative record of cell
shortening from this experiment; after administration of emigrated
neutrophils, the unloaded cell shortening decreased by 
50% (from
10% to 5% cell shortening) within 5 minutes. This represents
a very profound alteration in myocyte function. The cumulative data are
shown in Figure 3. Control unloaded cell
shortening in myocytes (not exposed to neutrophils) was 10.06±1.16%.
When neutrophils were added to the myocytes, a reduction of 50% in
unloaded cell shortening was observed (P<0.05). Addition of
anti-CD18 Ab did not protect the myocyte from the negative inotropic
effect of emigrated neutrophils (unloaded cell shortening at
6.96±1.15%, not significantly different from PMN group). The
anti-4 Ab, however, greatly reduced
neutrophil-induced impairment of cell shortening at 5 minutes
(9.42±0.94%, P<0.05). A similar pattern of results was
observed 10 minutes after the addition of emigrated neutrophils (data
not shown).
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Analysis of the data within each group by considering only
those myocytes that had adherent neutrophils, compared with those
without adherent neutrophils, demonstrated the importance of adherence
via the 4 integrin (Figure 4). In the group that received
neutrophils only (no Ab), only 1 myocyte out of 9 experiments had no
adherent neutrophils, and this cell did not show any change in unloaded
cell shortening (Figure 4A, left panel). This confirms previous
studies suggesting the absolute requirement of adherence in
neutrophil-mediated myocyte dysfunction. Of the remaining cells, 5
myocytes survived to the end of the experiment, and these had adherent
neutrophils ranging from 1 to 8 per myocyte. A negative inotropic
effect was measured in all of these cells at 5 minutes and all but 1
cell at 10 minutes (Figure 4A, right panel). It is noteworthy
that there was no correlation between the number of adherent
neutrophils and the amount of cellular dysfunction, inasmuch as a
single neutrophil was apparently able to induce similar amounts of
myocyte dysfunction as 8 neutrophils. Finally, 3 myocytes in this group
went into contracture and died within 5 minutes of neutrophil exposure
(Table 1), and they had 1, 2, and 4
adherent neutrophils, further emphasizing the ability of as few as 1
adherent neutrophil to induce myocyte dysfunction.
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In the anti-CD18 group (Figure 4B
, left panel), 2 myocytes did
not have any adherent neutrophils, and they showed no significant
decrease in unloaded cell shortening. One of these cells showed a
17.6% decrease from baseline at 5 minutes, but this cell completely
recovered by 10 minutes. However, in 6 of 7 cells that had adherent
neutrophils, there was a decrease in unloaded cell shortening despite
the presence of anti-CD18 Ab. These findings suggest, for the first
time, that immunoneutralization of CD18 is not sufficient to prevent
myocyte dysfunction in the presence of emigrated neutrophils. Finally,
2 of the myocytes in this group, which had 1 and 3 adherent
neutrophils, went into contracture within the first 5 minutes and died
(Table 1).
Importantly, in the anti-4 Ab group, 6 of the
8 myocytes had no adherent neutrophils, and the majority of these
myocytes showed no significant change in contractile activity (unloaded
cell shortening at 5 minutes), although 2 of these cells showed a
decline at 10 minutes. In this group, it was very difficult to find any
myocytes that supported neutrophil adhesion. In the 2 myocytes that did
have adherent neutrophils (1 and 4 neutrophils), there was a 19%
decrease in unloaded cell shortening in the former at 5 minutes, but
this cell completely recovered by 10 minutes. In the myocyte with 4
adherent neutrophils, there was no impairment in unloaded cell
shortening at either time point. In the group receiving
anti-4 Ab, no myocytes went into contracture
or failed to respond to the stimulus during the experiment (Table 1).
Myocyte function for all groups is summarized in Table 1.
Without any neutrophils added, all myocytes remained viable for the
entire experimental protocol, and none had any signs of dysrhythmia or
contractile dysfunction. When the emigrated neutrophils were added, 6
myocytes survived the protocol, but 4 of these exhibited dysrhythmia.
This abnormal activity included contractions independent of electrical
stimulation or a lack of, or delayed response to, electrical
stimulation. This phenomenon was also noted in 3 of 7 myocytes in the
group exposed to neutrophils in the presence of anti-CD18 Ab. In
contrast, none of the myocytes exposed to neutrophils in the presence
of anti-4 Ab behaved in this fashion.
Rates of contraction and relaxation for all groups are summarized in
Figure 5. The maximal rate of contraction
and relaxation did not change from baseline in the control group at 10
minutes (160.9±29.0 µm/second at baseline to 144.1±25.9
µm/second at 10 minutes for contraction rate, and 246.7±27.3 to
199.1±30.2 µm/second for relaxation rate, P=NS). The
addition of emigrated neutrophils, however, significantly reduced both
contraction and relaxation rates by 40% from baseline at 10 minutes
(P<0.05). The addition of either anti-CD18 or
anti-4 Ab protected the myocytes from this
neutrophil-induced decrease in contraction and relaxation. The fact
that the rate of contraction and relaxation was not reduced with
adherent neutrophils in the group that received Abs against either CD18
or 4 integrin suggests that the reduction in
contraction and relaxation rates observed in the neutrophil-alone group
is not a simple physical impedance of myocytes to contract due to
attached neutrophils. In the anti-CD18 group, the rate of contraction
changed from 150.84±21.0 to 143.78±11.2 µm/second, and the
rate of relaxation changed from 211.23±21.0 to 178.04±29.8
µm/second; P=NS. In the anti-4
group, the changes in rates of contraction and relaxation were
182.16±35.9 to 170.20±39.6 µm/second and 213.43±25.9 to
229.35±45.0 µm/second, respectively; P=NS.
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Figure 6 demonstrates that, unlike
emigrated neutrophils, circulating cells pretreated with 1% ZAP
adhered primarily via CD18. The addition of the
anti-4 Ab had no effect on the adhesion,
whereas the anti-CD18 Ab significantly decreased adhesion of
circulating neutrophils to myocytes by 40%. Further experiments
with these circulating cells showed that these cells could reduce
myocyte cell shortening by 35% from baseline at 5 and 10 minutes (n=2)
(myocytes with 1 or 2 adherent neutrophils) (Figure 7). Furthermore, the addition of
anti-CD18 Ab protected the myocyte (n=2) primarily through inhibition
of neutrophil adhesion (all experiments in the presence of anti-CD18 Ab
showed myocytes with no adherent neutrophils).
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Table 2 shows flow cytometry
fluorescence data for CD11b, CD18, and
4 integrin on circulating and emigrated
neutrophils. Mean fluorescence increased on emigration and
subsequent stimulation with ZAP for all 3 adhesion molecules
studied.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous work from our laboratory has shown that both CD18 and
4 integrin were essential for emigrated
neutrophil adherence to ventricular
myocytes.26 The present results extend this work and,
for the first time, suggest that engagement of the
4 integrin is critical for the ensuing myocyte
damage. In our study, the anti-CD18 Ab was able to protect the myocyte
from damage to mechanisms controlling contraction and relaxation rates
but was not able to protect against decreased cell shortening, myocyte
dysrhythmia, or contracture. This suggests that
4 integrin, not CD18, is the predominate
molecule in neutrophil-induced myocyte dysfunction. These observations
complement and significantly extend previous studies wherein
pretreatment with anti-CD18 Ab prevented neutrophil recruitment into
tissues.28 29 30 Clinically, patients arrive at hospital
after, not before, an infarct, at which point neutrophils have already
infiltrated the myocardium. Our data suggest that one could
therapeutically target the emigrated neutrophil to prevent ongoing
myocardial injury. Perhaps, both CD18 and 4
integrin pathways need to be inhibited to completely prevent
neutrophil-dependent injury in these
pathophysiological states wherein the
endothelium is injured by circulating neutrophils and
myocytes are injured by emigrated neutrophils.
Previous reports have described the ability of integrins to receive
signals from outside the cell that can, in turn, signal the release of
cytotoxic mediators from within the cell.31 Indeed,
engagement of CD18 on neutrophils leads to reorganization of the
cytoskeleton, oscillating cytosolic free calcium levels, shape
change, and subsequent secretion of granule proteins and
oxidants.32 33 This type of outside-in signaling can also
be mediated by the 4 integrin. Signal
transduction through the 4 integrin
activates protein tyrosine kinase activity in T
cells,34 and engagement of this fibronectin receptor
induces gene expression of enzymes, including collagenase
and metalloproteinase stromelysin in fibroblasts.35 The
role of the 4 integrin in neutrophils has yet
to be explored. Because both adhesion pathways are engaged in emigrated
neutrophil adhesion to myocytes, one might expect that inhibition of
either CD18 or 4 integrin would lead to
protection. Our study would suggest that adherence of emigrated
neutrophils only minimally activated a CD18-dependent pathway
of injury.
CD18 is upregulated on the neutrophil in response to stimulants in the
vasculature, which allows for firm adhesion to the
endothelium and subsequent emigration.36
Because the CD18 integrin has already been exposed to a stimulus before
emigration, it has already engaged its ligand. It is conceivable that
CD18 can no longer respond to a stimulus after emigration and therefore
is much less effective in initiating the release of specific cytotoxins
from the neutrophil. Although it is conceivable that CD18 could also be
reinternalized or shed from the surface of neutrophils after
emigration,37 our data do not support this view, as CD11b
and CD18 levels were increased on emigrated neutrophils (Table 2). In this study, and in previous studies, the
4 integrin is expressed at only very low
levels on circulating neutrophils, and this expression level is
increased after emigration and stimulation.26 38
Thereafter, 4 integrin is ready to engage its
receptor, and signaling via this ligand may be possible. The binding of
the 4 integrin to its ligand on the myocyte,
previously shown in the rat system to be fibronectin,26
can cause a release of proteases and oxidants from the neutrophil,
which can directly degrade the extracellular matrix. This may lead to
changes in membrane potential or integrity, thereby affecting the
availability of cystolic calcium and thus decreasing the
magnitude of cell shortening.
It is intriguing that the neutrophils appear to injure myocytes in a time- and site-specific manner. This is evidenced by the fact that global dysfunction did not occur in individual myocytes at the same time periods. Although we observed a very profound decrease in cell shortening at 5 minutes after neutrophil exposure with no Ab, we did not see a change in rate of contraction or relaxation until 10 minutes. These results suggest that the emigrated neutrophil was able to reduce cell shortening, before impacting on contraction or relaxation mechanisms. It is well appreciated that the degree of contraction, the rate of contraction, and the rate of relaxation are all mediated by different ionic events. This raises the possibility that the myriad of molecules released by the neutrophil impacts on ion channels with differing degrees of efficiency. Furthermore, it is possible that the neutrophil-induced damage is initially restricted to the sarcolemma, affecting L-type calcium channels, which trigger contraction by initiating a large calcium release from the sarcoplasmic reticulum (SR). Neutrophils may subsequently cause membrane depolarization, which could further reduce calcium influx via L-type calcium channels. At later times, intracellular organelles essential for excitation-contraction coupling and calcium homeostasis may be compromised. A decrease in calcium-induced calcium release from the SR results in a decreased rate of contraction. Moreover, the calcium pump in the SR and the rate of relaxation may also be significantly affected.
In conclusion, our results demonstrate that unlike circulating
neutrophils, emigrated neutrophils use 4
integrin to mediate the myocyte damage induced by neutrophils. To date,
most studies have focused on the mechanisms by which neutrophils adhere
to the endothelium and infiltrate the
myocardium, with the goal of targeting this mechanism to
reduce the injury associated with
pathophysiological conditions such as myocardial
infarction. The time window of opportunity to intervene in the
recruitment process may be so brief, however, that therapy may only
work prophylactically (ie, patients already have
neutrophils in the myocardium on arrival at hospital). Our
results provide a novel basis for therapeutic intervention on the
neutrophil that one may target even after this leukocyte has reached
the myocardium. This approach has the potential to reduce
or prevent myocyte dysfunction without affecting neutrophil function in
the circulation.
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Acknowledgments |
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We gratefully acknowledge support from the Canadian Medical Research Council (MRC) group grant. W.R.G. is an Alberta Heritage Foundation for Medical Research (AHFMR) scientist, and P.K. is an AHFMR senior scholar and MRC scientist.
Received November 30, 1998; accepted March 18, 1999.
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