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1 Department of Pediatrics and the Cardiovascular Research Center, 2 Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908; and 3 Department of Pharmacology, Merck Research Laboratories, West Point, Pennsylvania 19486
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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A3 adenosine receptors (A3ARs) have been
implicated in regulating mast cell function and in cardioprotection
during ischemia-reperfusion injury. The physiological role of
A3ARs is unclear due to the lack of widely available
selective antagonists. Therefore, we examined mice with targeted gene
deletion of the A3AR together with pharmacological studies
to determine the role of A3ARs in myocardial
ischemia-reperfusion injury. We evaluated the functional response to 15-min global ischemia and 30-min reperfusion in
isovolumic Langendorff hearts from A3AR
/
and wild-type (A3AR+/+) mice. Loss of
contractile function during ischemia was unchanged, but
recovery of developed pressure in hearts after reperfusion was improved
in A3AR/ compared with wild-type hearts
(80 ± 3 vs. 51 ± 3% at 30 min). Tissue viability assessed
by efflux of lactate dehydrogenase was also improved in
A3AR/ hearts (4.5 ± 1 vs. 7.5 ± 1 U/g). The adenosine receptor antagonist BW-A1433 (50 µM) decreased
functional recovery following ischemia in
A3AR/ but not in wild-type hearts. We also
examined myocardial infarct size using an intact model with 30-min left
anterior descending coronary artery occlusion and 24-h reperfusion.
Infarct size was reduced by over 60% in
A3AR/ hearts. In summary, targeted deletion
of the A3AR improved functional recovery and tissue
viability during reperfusion following ischemia. These data
suggest that activation of A3ARs contributes to myocardial injury in this setting in the rodent. Since A3ARs are
thought to be present on resident mast cells in the rodent myocardium, we speculate that A3ARs may have proinflammatory actions
that mediate the deleterious effects of A3AR activation
during ischemia-reperfusion injury.
knockout; myocardial infarction; inflammation; cardiac protection
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ADENOSINE IS RAPIDLY RELEASED in large amounts during cardiac ischemia and is a potent mediator of endogenous protective functions in the heart (6, 16, 45). Previous studies have shown that adenosine or adenosine receptor (AR) agonists can delay the onset of contracture (17), improve postischemic contractile function, reduce the rate of ATP catabolism (16), attenuate myocardial stunning (30), and reduce infarct size (3, 24, 42-44). Adenosine exerts its function via G protein-coupled receptors classified into at least four receptor subtypes: A1, A2A, A2B, and A3. The A3AR is the most recently identified subtype and has been cloned in multiple species, including rats (27, 49), sheep (21), humans (35), rabbits (11), and dogs (2).
Evaluation of the physiological role of A3ARs in the heart and other target tissues has been hampered by 1) multiple AR subtypes in the same target tissue, 2) lack of selective antagonists, and 3) diverse receptor affinities (particularly to xanthine antagonists) across species. Activation of A3ARs in a variety of models has been shown to elicit mast cell degranulation and subsequent release of inflammatory mediators (8, 12, 32, 37) to either induce (14, 38) or reduce apoptosis (9, 47) and to exert preconditioning (1, 24, 33, 39). Myocardial ischemia-reperfusion has also been shown to elicit degranulation of cardiac mast cells that may contribute to myocardial dysfunction and preconditioning (for a review, see Ref. 19). A3ARs have more recently been implicated in mediating cardioprotection from myocardial ischemia-reperfusion injury (3, 14, 43, 44). Pretreatment with selective A3 agonists has resulted in protection from ischemia-reperfusion injury in isolated hearts (43), attenuation of myocardial stunning and infarct size in conscious rabbits (3), and attenuation of postischemic dysfunction through ATP-sensitive K+ channels (KATP) (40, 44). Furthermore, Jordan et al. (14) recently reported that activation of A3ARs with 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide attenuated reperfusion injury by decreasing neutrophil function in isolated rabbit hearts.
Although there is increasing evidence suggesting a protective role for
the A3AR during ischemia-reperfusion injury, a recent report by Guo et al. (10) demonstrated that targeted
deletion of A3AR confers resistance to myocardial
ischemia injury, but targeted deletion did not prevent early
preconditioning. These disparate findings suggest that the role of the
cardiac A3AR needs further clarification. Accordingly, the
goal of this study was to further determine the role that
A3ARs play in protecting the heart from
ischemia-reperfusion injury in both isovolumic Langendorff hearts and intact animal myocardial infarction experiments in A3AR/ hearts and wild-type
(A3AR+/+) littermate controls. Although the
A3AR has been implicated in preconditioning/cardioprotection, our results and those of Guo et al.
(10) indicate that A3AR/
hearts are protected functionally as well as from infarction associated
with ischemia-reperfusion injury.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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A3AR/ mouse model.
Homozygous A3AR/ mice and wild-type
A3AR+/+ littermate controls were kindly
provided by Merck Research Laboratories. Generation and initial
characterization of the A3AR/ mice have
been described in detail previously (35).
Langendorff-perfused mouse heart model.
Hearts were isolated from 20- to 24-wk-old male and female wild-type
(A3AR+/+, 28 ± 2 g body wt,
n = 12) and A3AR/ mice
(31 ± 2 g body wt, n = 13) with homozygous
deletion of the A3AR. Mice were anesthetized with 50 mg/kg
pentobarbital sodium administered intraperitoneally, a thoracotomy was
performed, and hearts were rapidly excised into heparinized ice-cold
perfusion buffer. The aorta was cannulated, and the hearts were
retrogradely perfused at a constant pressure of 80 mmHg with modified
Krebs buffer containing (in mM) 118 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 2.5 CaCl2, 1.2 Mg2SO4, 11 glucose, and 0.6 EDTA. Buffer was
equilibrated with 95% O2-5% CO2 at 37°C,
giving a pH of 7.4 and a PO2 of ~550 mmHg.
The left ventricle was vented with a small polyethylene apical drain,
and a fluid-filled balloon constructed of plastic film was inserted
into the left ventricle via the mitral valve. The balloon was connected
to a pressure transducer by a polyethylene tube permitting continuous
measurement of left ventricular pressure. Hearts were immersed in
perfusate in a water-jacketed bath maintained at 37°C, and the
ventricular balloon was inflated to yield a left ventricular diastolic
pressure of 3-5 mmHg. Coronary flow was continuously monitored via
a Doppler flow probe (Transonic Systems; Ithaca, NY) located in the
aortic perfusion line. Aortic and left ventricular developed pressure
were recorded on a MacLab data acquisition system (AD Instruments;
Castle Hill, Australia) connected to an Apple 7300/180 computer. The
ventricular pressure signal was digitally processed on-line (using
MacLab Chart 3.5.6, AD Instruments) to yield diastolic and systolic
pressures, heart rate, and the first derivative of pressure over
time (dP/dt).
/ and wild type;
n = 6 for both groups) were allowed to stabilize for 30 min at intrinsic heart rate. Baseline diastolic and systolic pressures,
heart rate, and coronary flow were recorded, and 15-min normothermic
global ischemia was initiated by closing the aortic cannula and
simultaneously bubbling the bathing perfusate with 95%
N2-5% CO2 to reduce O2. Analysis
of the bathing perfusate showed that pH was unchanged and
PO2 fell to ~40 mmHg. Following
ischemia, reperfusion was initiated by unclamping the aortic
cannula and discontinuing the nitrogen bubbling. To verify the role of
A3AR activation in this model of
ischemia-reperfusion, a separate group of experiments was
performed in which we predominately blocked A1- and
A3ARs and compared the responses between
A3AR/ and wild-type animals. In the
wild-type animals, blocking A1 receptors would result in
negative functional effects as seen in our previous work
(26). If we also blocked A3 receptors, the
overall effect would be worsened recovery (if A3 receptors were beneficial) and a blunted negative effect (if A3
receptors were detrimental). In A3AR/
hearts, only A1 receptors could be blocked and the result
should be detrimental. An antagonist that blocks A1- and
A3ARs is BW-A1433, which has been shown to block rat
A1 receptors with an inhibitory constant
(Ki) of ~15 nM and rat A3
receptors with a Ki of ~24 µM (12,
19). BW-A1433 (50 µM; a gift from Susan Daluge,
Glaxo-Wellcome; Research Triangle Park, NC) was infused for 10 min
before global ischemia at ~1% of coronary flow and resumed
after global ischemia for the duration of reperfusion
(n = 7 for A3AR/ and
n = 6 for wild-type hearts).
Affinity of BW-A1433 for mouse A3AR. To determine the affinity of BW-A1433 for mouse A3 receptors, first we cloned the mouse A3AR. To do this we prepared A3 cDNA by RT-PCR from mouse testes. The primers used were as follows: forward, TCCGCCTGAAGCTTTTCTGAGCCACCATGGAA- GC; and reverse, TGGCTCTGTATCTGTCAAGGTAACTAC- TCA.
The sequence of the PCR product was checked and the cDNA was transferred to the mammalian expression plasmid CLDN10B. The mouse A3 receptor was introduced into HEK-293 cells with Lipofectin (GIBCO-BRL). Stably transfected cells were identified by growth in 0.5 µg/ml G418 and maintained in 0.25 µg/ml. The affinity of BW-A1433 was assessed by competition for the binding of 125I-labeled N6-(p-aminobenzyl)adenosine (125I-ABA) to membranes prepared from transfected cells (12).Determination of myocardial tissue viability via lactate
dehydrogenase efflux.
To assess degree of cell death in isolated hearts, coronary effluent
was collected from the fluid-filled bath surrounding the heart for
quantitation of lactate dehydrogenase (LDH) content (28).
An initial sample was collected at the end of 30-min equilibration for
baseline content. On the onset of reperfusion, coronary effluent was
collected and pooled throughout the 30-min period. The total volume of
pooled effluent was measured, and duplicates of 1.5-ml aliquots were
taken and stored at 
20°C until analysis. An LDH enzymatic assay kit
(Sigma) was optimized for sensitivity of detection and
spectrophotometric analysis to determine the LDH concentration in each
sample. Total LDH efflux was calculated by multiplying the enzyme
concentration in the sample by the total volume of coronary effluent
for each heart. To correct for different heart sizes, these values were
normalized for wet heart weight and expressed as units per gram similar
to previously reported methods (5, 23, 46).
In vivo mouse model of myocardial infarction. Mice were anesthetized with pentobarbital sodium at an initial dose of 100 mg/kg ip. Animals were placed in a supine position on a heating pad (Fine Science Tools; Foster City, CA), and their paws and tails were taped to the operating table. The upper portion of the trachea was exposed through a midline incision in the neck, and an endotracheal tube (polyethylene tubing, PE-60) was inserted orally into the trachea. Artificial respiration was maintained with a SAR-830/P ventilator (IITC Life Science, Woodland Hills, CA) with 80% oxygen at a frequency of 100 strokes/min, and a tidal volume of 1.5-2.5 ml. After intubation, the midline skin incision in the neck was extended down to the xiphoid process. The left third and fourth ribs were exposed by bluntly dividing the left pectoris major muscle and the muscles beneath it. A parasternal incision was made to open the left pleural cavity by cutting the left third and fourth ribs and intercostal muscles with a cautery pen (General Medical, Richmond, VA). A 7-0 silk suture was passed underneath the left anterior descending (LAD) coronary artery at a level just below the left atrium. Myocardial ischemia was induced by tying the suture over a piece of polyethylene (PE)-60 tubing for a 30-min period. Significant electocardiogram changes, including widening of QRS and elevation of S-T segment (monitored with a MacLab/8S Bridge/Bio Amplifier; AD Instruments), and color changes of the region at risk were used to confirm successful occlusion of the LAD coronary artery. Reperfusion was initiated by untying the suture from the PE-60 tubing and then removing the tubing but leaving the suture in place. A volume of 1-1.5 ml of 5% dextrose was given intraperitoneally to replace fluids lost during the operation. Rectal temperature was monitored throughout the operation with a rectal probe (thermocouple thermometer, Barnant; Barrington, IL) and was maintained between 36.5 and 37.5°C.
Determination of myocardial infarct size via triphenyltetrazolium chloride staining. After excision, hearts were cannulated through the ascending aorta and sequentially perfused with 2-3 ml of 0.9% sodium chloride solution and 3-4 ml of 1.0% triphenyltetrazolium chloride (TTC) in phosphate buffer (pH = 7.4, 37°C). After TTC staining, the LAD coronary artery was reoccluded by tightening the suture left in the myocardium after infarction. Hearts were then perfused with 2-3 ml of 10% Phthalo blue to delineate the nonischemic tissue. Hearts were frozen and trimmed free of right ventricle and atria, and the left ventricle was cut into 5-7 transverse slices. Slices were then fixed in 10% neutral buffered formalin solution. Each slice was weighed and photographed from both sides under a low magnification microscope (Olympus SZX12; Olympus Optical) mounted with a high-resolution digital camera (DVC-1300 RGB Color; DVC, Austin, TX). The imaging system was controlled by a computer running Image Pro-Plus (version 4.0, Media Cybernetics, Silver Spring, MD). The images were then transferred to PhotoShop (Adobe), and the borders of the infarction, ischemic area (risk region), and nonischemic area were traced for both sides of each tissue slice. The sizes of the nonischemic area, the risk region, and the infarction area of each slice were calculated as a percentage of corresponding total area multiplied by the total weight of the slice.
Statistical analysis. All results are expressed as means ± SE. Functional parameters during ischemia-reperfusion were analyzed by multivariate ANOVA for repeated measures with Bonferroni's correction for multiple comparisons and Tukey's post hoc test. Comparisons between groups for total LDH efflux and myocardial infarct size was made using Student's t-test. Comparison between groups for final recovery of developed pressure was made using a two-way ANOVA with Bonferroni's correction for multiple comparisons and Tukey's post hoc test. Statistical significance was accepted for P < 0.05.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Baseline function.
Baseline functional data for wild-type (heart wt 138 ± 16 mg,
n = 12) and A3AR/ isolated
hearts (heart wt 148 ± 14 mg, n = 13) are shown
in Table 1. No differences in
preischemic functional parameters or coronary flow rates were
observed between wild-type and A3AR/ hearts
at baseline (Table 1).
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Ischemia-reperfusion in wild-type and
A3AR/ isolated hearts.
Global normothermic ischemia immediately reduced heart rate and
contractile function, with full arrest within 5 min. Time to
ischemic contracture was 13.0 ± 1.9 min in wild-type
hearts and 13.1 ± 2.2 min in A3AR/
hearts. Diastolic pressure increased during ischemia in both wild-type and A3AR/ hearts to peaks of
40 ± 7 and 38 ± 10 mmHg, respectively. Heart rate and
coronary flow were comparable among all the study groups (Fig.
1, A and B).
Reactive hyperemia was initially observed on reperfusion in all groups,
which then returned to baseline by the end of reperfusion. (Fig.
1B). Myocardial function (expressed as the percent change
from baseline developed pressure) during ischemia-reperfusion
is shown in Fig. 2. With the onset of
reperfusion, hearts resumed spontaneous contraction within 30-60 s
and exhibited a marked recovery of left ventricular developed pressure.
After an initial increased recovery, there was a decline in function followed by a progressive recovery of developed pressure for the remainder of reperfusion. Deletion of the A3AR
(A3AR/) resulted in a better recovery of
left ventricular developed pressure compared with wild-type hearts at
all time points during reperfusion. These differences were particularly
evident when final recovery of left ventricular developed pressure was
examined, with A3AR/ hearts demonstrating
significantly better recovery of function compared with wild-type
hearts (67 ± 5 vs. 45 ± 2% of baseline, P < 0.05, Figs. 2 and 3). Cell death was estimated by total LDH efflux
in isolated hearts. Total LDH efflux was calculated to be 7.5 ± 0.9 U/g in wild-type hearts, whereas A3AR/
significantly reduced total efflux of LDH by ~40% to 4.5 ± 1.0 U/g (P < 0.05), providing evidence for improved tissue
viability along with the preserved function in
A3AR/ hearts.
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Affinity of BW-A1433 to mouse A3ARs.
Binding of 125I-ABA and BW-A1433 to recombinant mouse
A3ARs is shown in Fig. 4. Each triplicate assay contained
membrane protein, radioligand, and the indicated concentration of
BW-A1433. Figure 4, inset, shows equilibrium specific
binding of 125I-ABA to the same membrane proteins. The
maximum number of bound receptors (Bmax) of
125I-ABA was calculated to be 178 fmol/mg of protein and
the dissociation constant (Kd) was calculated to
be 6.9 nM. The Ki for BW-A1433 at mouse
A3 receptors was calculated to be 12.5 ± 4.5 µM. At
a concentration of 50 µM, BW-A1433 is predicted to occupy >80% of mouse A3 receptors and to decrease the potency of adenosine
by fivefold.
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Adenosine antagonism in wild-type and
A3AR/ hearts.
Administration of the nonselective antagonist BW-A1433 (50 µM) had no
significant effect on baseline functional parameters or coronary flow
in either wild-type or A3AR/ isolated
hearts (Table 1 and Fig. 1, A and B). The effect
of BW-A1433 on the time course and final recovery of left ventricular developed pressure following ischemia-reperfusion is shown in Figs. 2 and 3. Pretreatment with BW-A1433 resulted in no demonstrable change in recovery of left ventricular developed pressure at any time
point (Fig. 2), including final recovery of left ventricular developed
pressure following ischemia-reperfusion in wild-type hearts
(45 ± 2 vs. 42 ± 3% in treated hearts, Fig.
3). Conversely, A3AR/ hearts treated with the AR antagonist
demonstrated significantly reduced recovery of left ventricular
function to levels that approximated wild-type at all time points (Fig.
2). This was particularly evident when examining final recovery of left
ventricular developed pressure, which decreased from 67 ± 5 to 47 ± 4% of baseline with BW-A1433 treatment (Fig. 3).
Myocardial infarction in wild-type and
A3AR/ hearts.
To investigate the role of the A3AR in an intact animal
model of myocardial infarction, wild-type (n = 8) and
A3AR/ (n = 10) mice
underwent 30-min occlusion of the LAD coronary artery followed by
24 h of reperfusion. Infarct size (Fig. 5) and the area at risk
were determined by TTC staining and Phthalo blue staining,
respectively. There was no difference in the area at risk between the
two groups (39.6 ± 3.0 vs. 37.5 ± 2.0, P > 0.05). Targeted deletion of the A3AR resulted in a marked
reduction in myocardial infarct size (expressed as a percentage of the
area at risk) compared with wild-type mice, decreasing over 60% from 45 ± 5% to 13 ± 3% (P < 0.05) (Fig. 5).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, we examined the role of the A3AR in providing protection from ischemia-reperfusion injury in the mouse myocardium. For these studies we examined functional responses in Langendorff-perfused hearts and myocardial infarct size in an intact animal model of myocardial infarction. Moreover, we attempted to delineate the role of the A3AR in these studies by using an AR antagonist in a subset of isolated perfused hearts. Here, we present evidence that the A3AR plays a deleterious role during acute ischemia-reperfusion injury on both myocardial function and tissue viability in rodents by a mechanism that supercedes possible A3-mediated cardioprotection. Although a number of studies have examined the role A3ARs play in both ischemia-reperfusion injury and in ischemic preconditioning, the majority of these studies have been in models of either cell culture (cardiomyocytes) or isolated hearts utilizing exogenously applied A3 agonists (1, 14, 24, 40, 43, 44). However, the exact mechanisms associated with the observed cardioprotection afforded by A3ARs, particularly the "end-effector" in these models, are still not clearly defined. This lack of universal agreement on the physiological and functional role of A3ARs in the heart is primarily due to the diversity of both the cellular location and pharmacological profiles of these receptors across species. Although there are a number of specific agonists and antagonists of human, rabbit, and dog A3ARs, there are no known specific antagonists to rodent A3 receptors, with the possible exception of MRS-1523, which may block A3ARs in rats but has not yet been characterized in mice (9). This presents a challenge to the full investigation and comprehensive characterization of the role of the A3 receptor in these species. We therefore utilized a model with heterologous deletion of the A3 receptor in the mouse myocardium to assess its role in acute ischemia-reperfusion injury.
Our initial studies investigated the functional response to
ischemia-reperfusion injury in isolated Langendorff-perfused
wild-type and A3AR/ hearts. Deletion of
A3ARs did not effect baseline heart rate, end diastolic
pressure, coronary flow, or left ventricular developed pressure. This
is consistent with the recently published characterization of this
A3AR/ mouse model (35).
Contrary to our original hypothesis, functional recovery from
ischemia-reperfusion in A3AR/
hearts was unexpectedly improved compared with wild-type hearts. Recovery of left ventricular developed pressure (percentage of baseline) was significantly increased at all time points throughout reperfusion (Fig. 2), improving by ~50% by the end of 30-min
reperfusion. Interestingly, the A3AR/
hearts demonstrated no differences in either time to ischemic contracture or in peak diastolic pressure during ischemia
compared with wild-type hearts. Thus the beneficial effects of deleting the A3 receptor in the mouse myocardium appears to only
play a significant role during the reperfusion phase of
ischemia-reperfusion injury. This effect is consistent with the
notion of A3ARs triggering mast cell degranulation
(19) and a role for mast cells in reperfusion injury and
the inflammatory cascades that follow (8, 32). Total
efflux of LDH during reperfusion was reduced in
A3AR/ hearts compared with wild-type
hearts, which is an indication that these hearts had less cell death
and, therefore, demonstrated an improvement in tissue viability.
To confirm that the cardioprotection observed in these
A3AR/ mice is due to deletion of the
A3AR, we examined the effect of the AR antagonist BW-A1433
(50 µM) administered before ischemia and during reperfusion.
We chose this antagonist because high concentrations (>25 µM) have
been shown to block rat A3 receptors and to block
A3 receptor-mediated degranulation of rodent mast cells
(12). Furthermore, in the current study, we present data from 125I-ABA binding studies at an antagonist
concentration of 50 µM, which is predicted to block ~80% of the
mouse A3ARs (Fig. 4). It has
been well established by us and others that blocking the adenosine
A1 receptor decreases functional recovery in hearts subjected to ischemia-reperfusion injury (17,
28). On the other hand, our current results with
A3AR/hearts suggest that blocking the
A3AR might have a beneficial effect. Therefore, given the
known detrimental effect for A1 blockade and the observed
protective effect of A3 deletion, blocking both receptors
by treating wild-type hearts with BW-A1433 may be expected to have
opposing effects, whereas selective blockade of adenosine A1 receptors by treating A3AR/
hearts with BW-A1433 would be expected to adversely affect myocardial function. Baseline function in the BW-A4133-treated groups was similar
to the untreated groups, and they demonstrated similar functional time
course profiles (Fig. 2). On examination of final functional recovery
of left ventricular developed pressure (Fig. 3), wild-type hearts
treated with BW-A1433 showed no difference in functional recovery,
whereas A3AR/ hearts treated with BW-A1433
showed a significant decline in final functional recovery by ~30%.
These data are consistent with our proposed notion, that blockade of
A1 and A3 receptors may have opposing effects.
Furthermore, because BW-A1433 at the concentration used in this study
is a nonselective antagonist of all four AR subtypes, we reasoned that
the differential effects of BW-A1433 on wild-type and
A3AR/ mice was due to blockade of the
A3 receptor in wild-type mice. Another possibility in this
regard is whether there is an effect of
A3AR/ on A1AR expression.
Salvatore et al. (35) and Zhao et al. (48), in their initial characterization of this mouse, found no changes in
expression. Furthermore, Guo et al. (10) recently
confirmed this earlier finding: that there is no change to
A1 receptor expression in mice with the A3AR
deleted. Although these results are challenging to interpret, this
study does provide evidence that the increased functional recovery of
rodent hearts in response to ischemia-reperfusion injury in
this model appears to be a result of deletion of the A3AR.
To further examine this interesting improvement in functional recovery
and tissue viability in hearts lacking the A3AR, we examined A3AR/ mice in an intact animal
model of myocardial infarction. Assessment of myocardial infarct size
was used as the determinant of recovery (Fig.
5). Myocardial infarct size (expressed as
a percentage of the area at risk) in the
A3AR/ mice was markedly reduced by over
60%. These results add to the isolated heart functional data and
tissue viability data in providing evidence that activation of the
A3AR is deleterious both in vitro and in vivo. The data
from this study are consistent with and confirm the recently published
data by Guo et al. (10), who reported a decrease of
~35% in infarct size when the A3 receptor was deleted
(57.0 ± 2.9% in A3AR+/+ mice vs.
36.0 ± 4.0% in A3AR/).
Another important finding of this study is that deletion of the
A3AR appears to afford its protection during the
reperfusion phase of ischemia-reperfusion injury given there
was no difference in time to ischemic contracture or diastolic
dysfunction during ischemia, but rather an improvement in
recovery during the reperfusion period. These are interesting results
given the current interest in the possible role of inflammation in
ischemia-reperfusion injury. Adenosine has been reported to
potentiate inflammatory mediator release from a variety of mast cell
types including human lung mast cells (29), rat mast cells
(4, 12, 37), murine bone marrow mast cells
(24), and the rat RBL-2H3 cell line (30). More recent studies have demonstrated that the A3AR
promotes degranulation of rat mast cells both in vitro and in vivo
(32). Moreover, A3AR/ mice
have been used to provide further evidence that the A3AR mediates mast cell degranulation in murine bone marrow mast cells in
response to antigen (35, 41). Because mast cells are known to degranulate during ischemia-reperfusion injury
(13), we speculate that the protection observed in the
A3AR/ mice might be due to a lack of
A3-mediated mast cell degranulation and subsequent
inflammatory cascades. In support of the involvement of inflammatory
cascades, Guo et al. (10) recently published that
neutrophil infiltration within the infarcted region was less in the
A3AR/ mouse.
Therefore, if the lack of A3-mediated mast cell degranulation and subsequent release of inflammatory mediators is indeed the explanation of our observations, then this suggests a particularly important role for resident mast cells in the rodent heart because protection was observed in both isolated perfused hearts that lack circulating inflammatory cells as well as in our intact animal experiments. It is important to note that this A3-mediated response appears to be limited to the rodent model. In the human, it is the A2bAR subtype that has been reported to be present on mast cells and to stimulate mast cells (7, 22, 34). Although there may be limitations in extrapolating data from the current model to the human setting, an understanding of the A3 receptor on mast cells in rodents will help to identify the potential pathways, in both rodent and human alike, that are involved in ischemia-reperfusion injury and inflammatory responses.
In summary, the studies reported here are consistent with a role for the A3AR in an inflammatory cascade that is initiated as a result of ischemia-reperfusion injury in the rodent heart. We speculate that A3ARs may be acting deleteriously in both the in vitro and in vivo models of ischemia-reperfusion injury by triggering such an inflammatory pathway either by direct stimulation of mast cell degranulation with subsequent release of injurious mediators or via an as yet unknown mechanism. These results imply an important physiological role in mice for the A3AR in modifying both the functional outcome and tissue viability of the myocardium after ischemia-reperfusion injury. Furthermore, these data suggest that the view that activation of A3ARs is cardioprotective in the rodent heart following ischemia-reperfusion injury needs to be reevaluated.
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ACKNOWLEDGEMENTS |
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We thank Melissa Marshall for technical assistance with the mouse A3 cDNA and BW-A1433 affinity data.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-59419. G. P. Matherne was the recipient of an American Heart Association Established Investigator grant.
Address for reprint requests and other correspondence: G. P. Matherne, Dept. of Pediatrics, Univ. of Virginia Health Sciences Center, MR-4 Bldg., Box 801356, Charlottesville, VA 22908 (E-mail: gpm2y{at}virginia.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.
Received 24 January 2001; accepted in final form 5 July 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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P < 0.05, treated vs. untreated hearts.
