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Am J Physiol Heart Circ Physiol 292: H3164-H3171, 2007. First published February 16, 2007; doi:10.1152/ajpheart.00743.2005
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Cardioprotection by adenosine A2A agonists in a canine model of myocardial stunning produced by multiple episodes of transient ischemia

David K. Glover,1 Mirta Ruiz,1 Kazuya Takehana,1 Frank D. Petruzella,1 Jayson M. Rieger,2 Timothy L. Macdonald,2 Denny D. Watson,1 Joel Linden,1 and George A. Beller1

1Experimental Cardiology Laboratory, Cardiovascular Division, Department of Medicine, University of Virginia Health System, and 2Department of Chemistry, University of Virginia, Charlottesville, Virginia

Submitted 13 July 2005 ; accepted in final form 13 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
We sought to determine whether administration of a very low, nonvasodilating dose of a highly selective adenosine A2A receptor agonist (ATL-193 or ATL-146e) would be cardioprotective in a canine model of myocardial stunning produced by multiple episodes of transient ischemia. Twenty-four anesthetized open-chest dogs underwent either 4 (n = 12) or 10 cycles (n = 12) of 5-min left anterior descending coronary artery (LAD) occlusions interspersed by 5 or 10 min of reperfusion. Left ventricular thickening was measured from baseline through 180 min after the last occlusion-reperfusion cycle. Regional flow was measured with microspheres. In 12 of 24 dogs, A2A receptor agonist was infused intravenously beginning 2 min prior to the first occlusion and continuing throughout reperfusion at a dose below that which produces vasodilatation (0.01 µg·kg–1·min–1). Myocardial flow was similar between control and A2A receptor agonist-treated animals, confirming the absence of A2 receptor agonist-induced vasodilatation. During occlusion, there was severe dyskinesis with marked LAD zone thinning in all animals. After 180 min of reperfusion following the last cycle, significantly greater recovery of LAD zone thickening was observed in A2A receptor agonist-treated vs. control animals in both the 4-cycle (91 ± 7 vs. 56 ± 12%, respectively; P < 0.05) and the 10-cycle (65 ± 9 vs. 8 ± 16%, respectively; P < 0.05) occlusion groups. The striking amount of functional recovery observed with administration of low, nonvasodilating doses of adenosine A2A agonist ATL-193 or ATL-146e supports their further evaluation for the attenuation of postischemic stunning in the clinical setting.

left ventricular dysfunction; demand ischemia; reperfusion injury

MYOCARDIAL STUNNING IS A FORM of reversible reperfusion injury characterized by prolonged depression of left ventricular (LV) contractile function in viable myocardium despite restoration of normal or near-normal resting blood flow (7, 28). Clinical studies have demonstrated that many patients with coronary artery disease experience repetitive episodes of myocardial ischemia in the same vascular territory while going about their daily activities (14). Echocardiographic and radionuclide studies have demonstrated that severe postischemic contractile dysfunction, or myocardial stunning, occurs following periods of exercise or pharmacological stress-induced ischemia (15, 30, 34). Importantly, animal studies have shown that frequent episodes of myocardial ischemia that occur in close temporal proximity have a cumulative effect on the deterioration of myocardial contractility, and some (29, 33, 52) have proposed that repetitive myocardial stunning following demand ischemia is the mechanism for myocardial hibernation. In addition, this phenomenon has been proposed as one mechanism for regional systolic dysfunction in unstable coronary syndromes.

It is now thought that the major pathophysiological mechanism underlying myocardial stunning involves damage to myofibrillar and/or sarcoplasmic-reticular proteins by highly reactive oxygen species, particularly the superoxide anion (O–) and hydroxyl radical (bulletOH), that are produced in the first few minutes of reperfusion with resulting disruption in calcium homeostasis (4–6, 51). Increased cytokine production with ischemia-reperfusion might also play a pathogenic role in myocardial stunning.

Recent experimental evidence indicates that adenosine markedly attenuates stunning after a period of myocardial ischemia (37, 50). Furthermore, adenosine receptor blockade has been shown to worsen myocardial stunning, presumably by inhibiting the protective effects of endogenous adenosine produced during the ischemic period (9, 40). The mechanism for the adenosine-mediated protection against myocardial stunning is unknown; however, it does not involve preconditioning, because it has been shown to be protective even when given just before reperfusion (37, 50).

We hypothesized that the anti-inflammatory property of adenosine is at least partly responsible for the cardioprotection against myocardial stunning. Specifically, adenosine, acting on A2A receptors on the vascular endothelium and on all bone marrow-derived white cells and platelets, inhibits inflammatory processes, including neutrophil adhesion and extravasation and platelet aggregation. It also prevents the release of injurious cytokines, proteases, and reactive oxidative species such as O– and bulletOH. Accordingly, in the present study we tested this hypothesis by administering a very low, nonvasodilating dose of highly selective adenosine A2A (AA2A) receptor agonists in a canine model of myocardial stunning produced by repetitive episodes of brief coronary occlusion interspersed with reperfusion. Low doses of these AA2A receptor agonists have previously been shown to markedly reduce inflammation and to protect tissue in a number of clinically relevant animal models of ischemia-reperfusion injury (13, 22, 41, 56).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Surgical preparation. Twenty-four fasted, adult mongrel dogs (mean weight 25.6 ± 1.0 kg; range, 15.9–34.5 kg) were anesthetized with pentobarbital sodium (30 mg/kg), tracheally intubated, and mechanically ventilated with room air on a respirator (model 613; Harvard Apparatus) with positive end-expiratory pressure of 5 cm H2O. A left lateral thoracotomy was performed at the level of the fifth intercostal space, and the heart was suspended in a pericardial cradle. A proximal portion of the left anterior descending coronary artery (LAD) was dissected free of the epicardium and was loosely encircled with a snare occluder. Animals were instrumented as previously described for measurement and recording of heart rate (HR), mean arterial pressure (MAP), left atrial pressure, LAD, and left circumflex coronary artery (LCx) flows (model T-206; Transonic Systems). To measure LV pressure and its first time derivative (dP/dt), a high-fidelity pressure-recording catheter (Millar Instruments) was inserted into a carotid artery sheath and was advanced while observing the pressure tracing until its tip rested within the LV cavity. Regional systolic thickening was measured by the epicardial crystal pulsed-Doppler technique (model DMM-10; Crystal Biotech) that has been previously validated in the canine model and has been used extensively by our group (11, 22, 60).

The femoral arteries were cannulated with 8-Fr polyethylene catheters for arterial blood gas monitoring (model 170; CIBA-Corning) and for arterial reference blood withdrawals for microsphere determination of regional blood flows (11, 22, 23, 27). The core temperature of the animal was held constant throughout each experiment by using a thermostatically controlled heat lamp with a set point equal to the initial core temperature of the animal prior to opening the chest cavity.

The investigation conformed with the "Guiding Principles in the Care and Use of Animals," published by the American Physiological Society, and all experiments were performed with the approval of the Institutional Animal Care and Use Committee at the University of Virginia.

Experimental protocol. After instrumentation of the animals, steady-state baseline hemodynamic and wall-thickening measurements were made and regional myocardial blood flow was assessed with a left atrial injection of 2.5 x 106 radiolabeled microspheres (either Cr51, Sr85, Nb95, or Sc46; 15 µm diameter). The microsphere technique is routinely used in our laboratory for measuring regional myocardial blood flow and has been previously described in detail (11, 22, 23, 27).

Stock solutions of the highly selective A2A agonist compounds ATL-193 (5 mg/ml; mol wt = 500.55) and ATL-146e (4.86 mg/ml; mol wt = 486.2) were previously prepared in 100% DMSO. On the day of each experiment, a dog weight-adjusted aliquot of the stock (range 9.6–21.3 µl) was diluted in 120 ml saline, giving a final concentration ranging from 0.40 to 0.86 µg/ml for the infusion solution. The cumulative dose of DMSO administered in these dogs was <0.5 µl/kg body wt.

In 12 dogs (group 1), mild-to-moderate myocardial stunning was produced by four cycles of transient LAD occlusion consisting of 5-min occlusion periods interspersed by 10-min reperfusion periods. Six of these twelve dogs received a treatment consisting of a low-dose intravenous infusion (0.01 µg·kg–1·min–1) of the AA2A receptor agonist ATL-193 (23, 45) beginning 2 min prior to the first occlusion and continuing throughout the entire reperfusion period, whereas the remaining six control dogs received an intravenous infusion over the same period of time of an equivalent volume of saline (~97 ml).

In a second group of 12 dogs (group 2), a more severe model of myocardial stunning was used by performing 10 cycles of transient LAD occlusion (5 min occlusion, 5 min reperfusion). As in group 1, 6 of the 12 group 2 dogs were treated with a low-dose intravenous infusion (0.01 µg·kg–1·min–1) of ATL-193 (n = 2) or ATL-146e (n = 4) and 6 control dogs received an intravenous infusion of an equivalent volume of saline (~113 ml) over the same time period. Both ATL-193 and ATL-146e have similar potency and very high selectivity for the A2A receptor subtype (45), and both compounds have been shown to produce similar hemodynamic effects at high, vasodilating doses in our laboratory (23). The use of one of the A2A agonists versus the other was based on the availability of the compounds at the time when these studies were being conducted.

At the end of the experimental protocol, the dogs were euthanized with an overdose of pentobarbital sodium and potassium chloride, and the heart was rapidly excised. The heart was then sliced into four rings from apex to base and subdivided into 72~1.0-g segments. Regional myocardial blood flow (ml·min–1·g–1) in each segment was quantified by gamma-well counting as previously described in detail (11, 22, 23, 27). Segments with endocardial flow of ≤0.3 ml·min–1·g–1 during LAD occlusion were classified as central LAD zone, whereas those segments with endocardial flow of >0.5 ml·min–1·g–1 were classified as LCx zone.

Statistical analysis. All statistical computations were made with SYSTAT software. Differences between groups over time were assessed by two-way repeated-measures ANOVA followed by Bonferroni's post hoc testing when multiple comparisons were made. Single paired comparisons within a group were made by using a paired t-test. The results were expressed as means ± SE. Probability values <0.05 were considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 DISCLOSURES
 REFERENCES
 
Hemodynamic data. Mean hemodynamic data are summarized in Tables 1 and 2. In both group 1 (4 cycles) and group 2 (10 cycles), there were no significant variations over time in HR, MAP, or dP/dt, irrespective of the absence or presence of the AA2A receptor agonist. Also, in both groups of dogs left atrial pressure increased slightly during occlusion and tended to return to baseline values afterward. In group 1 and group 2 dogs, ultrasonic flow decreased during each LAD occlusion.


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  		Table 1. Hemodynamic parameters, group 1

 

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  		Table 2. Hemodynamic parameters, group 2

 
In the group 2 dogs that underwent 10 occlusion-reperfusion cycles, reactive hyperemia was present in the reperfused LAD zone 5 min after the last cycle in both the control and AA2A receptor agonist-treated subgroups but resolved quickly.

There was no evidence of vasodilatation caused by the AA2A receptor agonist infusion in any of the treated dogs in group 1 or 2.

Regional myocardial blood flow. Regional myocardial blood flow data are presented in Tables 3 and 4. Overall, there were no significant differences in regional flows between control and AA2A receptor agonist-treated animals in either group of dogs, with the exception of a small increase in endocardial flow in the normal zone of group 1 dogs at 180 min after reperfusion. In all dogs in both groups, regional flow in the endocardial, midwall, and epicardial layers of the LAD zone decreased significantly during occlusion, with endocardial flow being <0.15 ml·min–1·g–1. In all dogs in both groups, LAD zone flow returned to baseline following the last occlusion-reperfusion cycle. With the one exception noted above, normal LCx zone flow was not significantly different from baseline at any time point, confirming the absence of vasodilatation with the AA2A agonist infusion at the low dose administered (0.01 µg·kg–1·min–1).


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  		Table 3. Regional myocardial blood flow, group 1

 

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  		Table 4. Regional myocardial blood flow, group 2

 
Regional myocardial systolic thickening. Normal LCx zone regional systolic thickening is shown in Table 5. Note that in both groups of dogs, regional systolic thickening in the normal zone was similar in the presence or absence of AA2A agonist treatment at all time points, confirming that there was no positive inotropic effect of the AA2A agonist compounds at the low doses administered in this study.


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  		Table 5. Normal LCx zone regional systolic thickening

 
Regional systolic thickening data from the occluded-reperfused LAD zones of group 1 and group 2 dogs are presented in Figs. 1 and 2, respectively. In animals subjected to four cycles of coronary occlusion and reperfusion (group 1), the LAD zone was dyskinetic during each occlusion cycle, as shown by negative thickening values. In control animals (Fig. 1A), regional thickening was decreased by 65% immediately after the last occlusion-reperfusion cycle and remained significantly inhibited by ~45% for the next 3 h.


Figure 1
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  		Fig. 1. Regional myocardial percent systolic thickening in animals subjected to 4 cycles of 5-min left anterior descending coronary artery (LAD) occlusion (O) interspersed by 10 min of reperfusion (R) in the absence (A) or presence (B) of an intravenous infusion of a low-dose (0.01 µg·kg–1·min–1) of an AA2A receptor agonist (ATL). Values in each subgroup are means ± SE of 6 experiments. *P < 0.05 vs. baseline, {dagger}P < 0.05 vs. control.

 

Figure 2
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  		Fig. 2. Regional myocardial percent systolic thickening in animals subjected to 10 cycles of 5-min LAD occlusion (O) interspersed by 5 min of reperfusion (R) in the absence (A) or presence (B) of an intravenous infusion of a low dose (0.01 µg·kg–1·min–1) of an AA2A receptor agonist (ATL). Values in each subgroup are means ± SE of 6 experiments. *P < 0.05 vs. baseline, {dagger}P < 0.05 vs. control.

 
In the presence of AA2A agonist (Fig. 1B), systolic thickening was decreased by 43% immediately after the last coronary reperfusion and full recovery of function was observed as soon as 45 min after reperfusion (P = not significant vs. baseline).

After 10 cycles of coronary occlusion and reperfusion (group 2), the LAD zone was akinetic in control dogs, as shown by a mean thickening value decreased by 88% when compared with baseline, and no improvement was observed over the course of the next 3 h (Fig. 2A), indicative of severe stunning. Intravenous infusion of a low dose of AA2A agonist (Fig. 2B) resulted in a significant improvement as early as 5 min after the last occlusion-reperfusion cycle in treated dogs, with a 55% decrease when compared with baseline (P < 0.01 vs. control). After 3 h of reperfusion, LAD systolic thickening returned to 65% of the baseline value (P < 0.05 vs. control). Similar results were obtained by using either ATL-193 or ATL-146e in this group.

Recovery of LV function in the LAD zone following either 4 or 10 cycles of transient ischemia is summarized in Fig. 3. As shown, there was significantly greater recovery of thickening after reperfusion with AA2A agonist in both the 4- (56 vs. 91%) and 10-cycle (8 vs. 65%) groups. Note that this greater amount of recovery in the AA2A agonist-treated dogs was observed as early as 5 min after the 10th occlusion cycle.


Figure 3
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  		Fig. 3. Recovery of LAD zone thickening over time following the final occlusion-reperfusion cycle in both group 1 (4 cycles) and group 2 (10 cycles). In both groups, there was significantly greater recovery of left ventricular thickening in the presence of the AA2A receptor agonist treatment vs. control. Note that in the severe stunning group (group 2), there was significantly greater recovery as early as 5 min in the AA2A agonist-treated subgroup vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
In the present study we found that, in a canine model of moderate-to-severe myocardial stunning produced by multiple, brief episodes of transient ischemia, intravenous administration of a low dose of an AA2A receptor agonist (ATL-193 or ATL-146e) abolished or markedly attenuated the postischemic impairment in LV function. This is the first experimental study to demonstrate that this improvement in contractile function was unrelated to changes in coronary blood flow or other hemodynamic effects.

The exact mechanism by which the AA2A receptor agonist protected the myocardium against stunning in our study is unknown. One possibility is that the AA2A receptor agonist directly inhibited the production of oxygen free radicals that are known to play a major role in causing myocardial stunning (6). This concept is supported by experimental data from Jordan et al. (31) showing that another AA2A receptor agonist, CGS-21680, markedly reduced O– production in reperfused canine myocardium and inhibited neutrophil adherence and accumulation. Although neutrophils are no longer believed to be the major source of oxygen free radicals involved in myocardial stunning (3), the vascular endothelium itself can produce free radicals via the enzymes xanthine oxidase and NADH/NADPH oxidase. Because AA2A receptors are also located on the endothelium, it is quite possible that stimulation of these receptors may act directly on these enzymes to inhibit the production of free radicals.

A second possibility is that the oxygen free radical concentration was reduced secondary to an increased production of nitric oxide by the constitutive form of nitric oxide synthase in the endothelium (eNOS). The AA2A receptor agonist CGS-21680 has been shown to increase the production of nitric oxide by both human and porcine cultured arterial endothelial cells (38, 44), as well as in porcine microvessels (25). Although nitric oxide is itself a free radical and is cytotoxic at high concentrations, at lower concentrations it is also a free radical scavenger of O– and has been shown to be cytoprotective (1, 46). In a pig model of myocardial stunning, Engelman et al. (16) reported that intravenous administration of L-arginine, the nitric oxide precursor molecule, significantly reduced free radical-mediated lipid peroxidation, plasma levels of soluble adhesion molecules, myocardial stunning, and arrhythmias. Furthermore, studies in eNOS-knockout mice have clearly shown that deletion of eNOS exacerbates myocardial stunning (24), whereas overexpression of eNOS in cardiac myocytes was shown to be protective (8).

A third possibility is that AA2A receptor activation was protective by inhibiting the release of inflammatory cytokines and chemokines from tissue-resident mast cells, macrophages, or possibly even from the myocytes themselves. In addition to their role in initiating and amplifying postischemic inflammatory reactions, cytokines, particularly TNF-{alpha}, IL-6, and IL-1, can directly depress contractile function (55). Importantly, TNF-{alpha} can be measured in the coronary effluent from isolated hearts during ischemia-reperfusion and has been shown to contribute to postischemic contractile dysfunction (10, 26, 42). Furthermore, Cain et al. (10) demonstrated that adenosine reduced cardiac TNF-{alpha} production following reperfusion in isolated rat hearts and that TNF-{alpha}-binding protein confers protection similar to that of adenosine against stunning in human myocardium. One possible source of TNF-{alpha} in cardiac tissue is from tissue-resident mast cells and macrophages (18, 53). Frangogiannis et al. (18) demonstrated that tissue-resident cardiac mast cells degranulate following coronary reperfusion and release preformed TNF-{alpha} that eventually leads to the upregulation of IL-6. Importantly, both mast cells and macrophages are known to have functional AA2A receptors on their plasma membranes, and stimulation of these receptors inhibits these cells from degranulating (17).

In summary, although the exact mechanism for the reduction in myocardial stunning with the AA2A agonist treatment remains unknown, there are a number of ways in which these compounds can protect the heart by reducing the levels of injurious oxygen free radicals and by inhibiting the production and/or release of inflammatory cytokines with known negative inotropic properties. Further studies are required to address these mechanisms in detail.

Comparison with studies from other laboratories. Investigators from other laboratories (36, 39) have also shown a reduction in myocardial stunning by using the AA2A receptor agonist CGS-21680. In pigs subjected to myocardial ischemia (15 min LAD occlusion) and reperfusion, Lasley et al. (36) administered CGS-21680 directly into the coronary artery beginning 120 min after reperfusion and found that myocardial contractility was improved in the reperfused zone. In this study, the functional improvement cannot be attributed to an inhibition of oxygen free radicals or an anti-inflammatory effect because the treatment did not begin until 2 h after reperfusion. Although coronary flow also increased, the authors ruled out the flow increase as the mechanism for the increased inotropy. Nevertheless, at least part of the positive inotropic effect observed with CGS-21680 in the Lasley study (36) may have been a systemic effect because, despite the fact that CGS-21680 was given via the intracoronary route, heart rate increased significantly. One possibility is that the increased contractility may have resulted from stimulation of AA2A receptors in the peripheral nervous system, including the baroreceptors and chemoreceptors in the carotid body (21, 32). Stimulation of these autonomic receptors by AA2A receptor agonists is excitatory, has been shown to enhance sympathetic drive (49), and would be expected to increase contractility. This potential mechanism is supported by their finding that the intracoronary infusion of CGS-21680 produced tachycardia without an accompanying reduction in MAP. Although CGS-21680 had no effect on contractility in normal myocardium, this might be explained by the fact that stunned myocardium is known to be hypersensitive (57), i.e., factors that affect contractility in normal myocardium have a greater impact in stunned myocardium (2). It is interesting that in another study, CGS-21680 had no cardioprotective effect in a similar canine model of stunning produced by multiple, brief episodes of coronary artery occlusion and reperfusion (58). Although it is possible that this result could be dose-related, it might also represent differences in the biodistribution between CGS-21680 and the A2A agonists ATL-146e or ATL-193 used in the present study. CGS-21680 is a carboxylic acid whose positive charge may hinder or prevent the molecule from leaving the intravascular space and reaching potential interstitial targets of action known to have AA2A receptors such as tissue-resident mast cells and/or macrophages.

Clinical implications. ATL-146e, one of the AA2A agonists used in this study, has already undergone safety testing and a Phase 2 clinical trial for high-dose bolus administration as a vasodilator for pharmacological stress myocardial perfusion imaging studies (Bristol-Myers Squibb Medical Imaging). The potential benefit of using a low dose of an AA2A agonist, such as ATL-146e, in the clinical setting to prevent or treat postischemic dysfunction secondary to stunning needs to be explored. The current experimental study in a canine model of repetitive stunning provides proof of principle that prolonged postischemic dysfunction can be significantly attenuated and recovery can be hastened by infusion of an AA2A agonist by using a dose that does not produce hypotension or other systemic hemodynamic changes. The striking amount of functional recovery observed with ATL-193 and ATL-146e supports their further evaluation for the attenuation of postischemic stunning after reperfusion therapy in patients. Also, if repetitive stunning is indeed a major pathophysiological mechanism in myocardial hibernation, a long-acting oral formulation of an AA2A agonist could prove to be beneficial for cardioprotection (12, 19, 35, 47, 48).

Study limitations. One limitation of our study was that it was conducted by using an open-chest, anesthetized canine model. Previous studies have shown that the magnitude of changes produced by the ischemic insult (i.e., free radical production, LV dysfunction) are greater in the anesthetized, open-chest model than observed under the same experimental conditions in conscious animals (57). In the present study, all dogs underwent the same experimental procedures and comparisons of LV function were made between the control and treated groups of dogs. If the oxygen free radical burst is indeed higher in anesthetized dogs, then this makes the amount of protection observed in this study all the more impressive.

A second limitation was that the AA2A receptor agonist was administered beginning prior to the first occlusion-reperfusion cycle. Thus it is difficult to ascertain whether the protection afforded by the treatment was due to a preconditioning effect, an anti-ischemic effect, or attenuation of reperfusion injury. As previously mentioned, we intentionally began the treatment prior to the first occlusion because prior studies have shown that a significant oxygen free radical burst occurs even after the first occlusion-reperfusion cycle, with additional and diminishing bursts occurring with subsequent cycles (6). It is unlikely that the antistunning effect observed in our study with ATL-146e was due to preconditioning or to an anti-ischemic effect. Although adenosine is known to be an important trigger of preconditioning, numerous investigators have shown that the preconditioning mechanism with adenosine involves stimulation of the adenosine A1 and possibly A3 receptor subtypes on cardiomyocytes (35). Furthermore, the improvement in postischemic dysfunction observed in our study was not likely due to an anti-ischemic effect of ATL-146e because coronary flow was not enhanced and systemic hemodynamics were unchanged.

A third limitation of this study was the fact that no postmortem tissue analysis was performed to document that the reperfused myocardium was not irreversibly injured by the repetitive occlusion and reperfusion cycles. However, this experimental model of stunning produced by multiple episodes of brief ischemia has been well documented in the literature in studies using the same or an even greater number of occlusion-reperfusion cycles of similar duration (6, 43, 54, 59). In these previous studies, there was a comparable degree of postischemic LV dysfunction with no evidence of myocardial necrosis by either postmortem triphenyltetrazolium chloride staining or electron microscopy in any experimental animal (6, 43, 54, 59).

Finally, it is impossible to determine from our study whether the mechanism for protection involves a reduction in the production of oxygen free radicals or an inhibition of the release of inflammatory cytokines such as TNF-{alpha} because we did not measure any of these parameters. Future studies using electron spin-trap techniques to measure free radical production and/or immunohistochemical techniques to measure cytokines in this model may provide an answer to these important questions.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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DISCLOSURES
 REFERENCES
 
This study was funded by an American Heart Association Grant-in-Aid no. 0050607N.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
The AA2A agonists, ATL-193 and ATL-146e, used in this study are owned by Adenosine Therapeutics. T. Macdonald, J. Linden, G. A. Beller, and D. K. Glover have equity positions in, and J. M. Rioger is an employee of, this company.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. K. Glover, Cardiovascular Division, Dept. of Medicine, P.O. Box 800500, Univ. of Virginia Health System, Charlottesville, VA 22908-0500 (e-mail: dglover{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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

  1. Bloodsworth A, O'Donnell VB, Freeman BA. Nitric oxide regulation of free radical- and enzyme-mediated lipid and lipoprotein oxidation. Arterioscler Thromb Vasc Biol 20: 1707–1715, 2000.[Abstract/Free Full Text]
  2. Bolli R. Common methodological problems and artifacts associated with studies of myocardial stunning in vivo. Basic Res Cardiol 90: 257–262, 1995.[CrossRef][Web of Science][Medline]
  3. Bolli R. Role of neutrophils in myocardial stunning after brief ischaemia: the end of a six year old controversy (1987–1993). Cardiovasc Res 27: 728–730, 1993.[Web of Science][Medline]
  4. Bolli R, Jeroudi MO, Patel BS, DuBose CM, Lai EK, Roberts R, McCay PB. Direct evidence that oxygen-derived free radicals contribute to postischemic myocardial dysfunction in the intact dog. Proc Natl Acad Sci USA 86: 4695–4699, 1989.[Abstract/Free Full Text]
  5. Bolli R, Marban E. Molecular and cellular mechanisms of myocardial stunning. Physiol Rev 79: 609–634, 1999.[Abstract/Free Full Text]
  6. Bolli R, Zughaib M, Li XY, Tang XL, Sun JZ, Triana JF, McCay PB. Recurrent ischemia in the canine heart causes recurrent bursts of free radical production that have a cumulative effect on contractile function. A pathophysiological basis for chronic myocardial "stunning". J Clin Invest 96: 1066–1084, 1995.[Web of Science][Medline]
  7. Braunwald E, Kloner RA. The stunned myocardium: prolonged, postischemic ventricular dysfunction. Circulation 66: 1146–1149, 1982.[Abstract/Free Full Text]
  8. Brunner F, Maier R, Andrew P, Wolkart G, Zechner R, Mayer B. Attenuation of myocardial ischemia/reperfusion injury in mice with myocyte-specific overexpression of endothelial nitric oxide synthase. Cardiovasc Res 57: 55–62, 2003.[Abstract/Free Full Text]
  9. Bunch FT, Thornton J, Cohen MV, Downey JM. Adenosine is an endogenous protectant against stunning during repetitive ischemic episodes in the heart. Am Heart J 124: 1440–1446, 1992.[CrossRef][Web of Science][Medline]
  10. Cain BS, Harken AH, Meldrum DR. Therapeutic strategies to reduce TNF-alpha mediated cardiac contractile depression following ischemia and reperfusion. J Mol Cell Cardiol 31: 931–947, 1999.[CrossRef][Web of Science][Medline]
  11. Calnon DA, Glover DK, Beller GA, Vanzetto G, Smith WH, Watson DD, Ruiz M. Effects of dobutamine stress on myocardial blood flow, 99mTc sestamibi uptake, and systolic wall thickening in the presence of coronary artery stenoses: implications for dobutamine stress testing. Circulation 96: 2353–2360, 1997.[Abstract/Free Full Text]
  12. Chen C, Ma L, Linfert DR, Lai T, Fallon JT, Gillam LD, Waters DD, Tsongalis GJ. Myocardial cell death and apoptosis in hibernating myocardium. J Am Coll Cardiol 30: 1407–1412, 1997.[Abstract]
  13. Day YJ, Huang L, McDuffie MJ, Rosin DL, Ye H, Chen JF, Schwarzschild MA, Fink JS, Linden J, Okusa MD. Renal protection from ischemia mediated by A2A adenosine receptors on bone marrow-derived cells. J Clin Invest 112: 883–891, 2003.[CrossRef][Web of Science][Medline]
  14. Deanfield JE, Selwyn AP. Character and causes of transient myocardial ischemia during daily life. Implications for treatment of patients with coronary disease. Am J Med 80: 18–24, 1986.[Web of Science][Medline]
  15. Druz RS, Akinboboye OA, Grimson R, Nichols KJ, Reichek N. Postischemic stunning after adenosine vasodilator stress. J Nucl Cardiol 11: 534–541, 2004.[CrossRef][Web of Science][Medline]
  16. Engelman DT, Watanabe M, Maulik N, Cordis GA, Engelman RM, Rousou JA, Flack JE 3rd, Deaton DW, Das DK. L-Arginine reduces endothelial inflammation and myocardial stunning during ischemia/reperfusion. Ann Thorac Surg 60: 1275–1281, 1995.[Abstract/Free Full Text]
  17. Fenster MS, Shepherd RK, Linden J, Duling BR. Activation of adenosine A2 alpha receptors inhibits mast cell degranulation and mast cell-dependent vasoconstriction. Microcirculation 7: 129–135, 2000.[CrossRef][Web of Science][Medline]
  18. Frangogiannis NG, Lindsey ML, Michael LH, Youker KA, Bressler RB, Mendoza LH, Spengler RN, Smith CW, Entman ML. Resident cardiac mast cells degranulate and release preformed TNF-alpha, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion. Circulation 98: 699–710, 1998.[Abstract/Free Full Text]
  19. Frangogiannis NG, Shimoni S, Chang SM, Ren G, Dewald O, Gersch C, Shan K, Aggeli C, Reardon M, Letsou GV, Espada R, Ramchandani M, Entman ML, Zoghbi WA. Active interstitial remodeling: an important process in the hibernating human myocardium. J Am Coll Cardiol 39: 1468–1474, 2002.[Abstract/Free Full Text]
  20. Gao WD, Liu Y, Marban E. Selective effects of oxygen free radicals on excitation-contraction coupling in ventricular muscle. Implications for the mechanism of stunned myocardium. Circulation 94: 2597–2604, 1996.[Abstract/Free Full Text]
  21. Gauda EB. Expression and localization of A2a and A1-adenosine receptor genes in the rat carotid body and petrosal ganglia. A2a and A1-adenosine receptor mRNAs in the rat carotid body. Adv Exp Med Biol 475: 549–558, 2000.[Web of Science][Medline]
  22. Glover DK, Riou LM, Ruiz M, Sullivan GW, Linden J, Rieger JM, Macdonald TL, Watson DD, Beller GA. Reduction of infarct size and postischemic inflammation from ATL-146e, a highly selective adenosine A2A receptor agonist, in reperfused canine myocardium. Am J Physiol Heart Circ Physiol 288: H1851–H1858, 2005.[Abstract/Free Full Text]
  23. Glover DK, Ruiz M, Takehana K, Petruzella FD, Riou LM, Rieger JM, Macdonald TL, Watson DD, Linden J, Beller GA. Pharmacological stress myocardial perfusion imaging with the potent and selective A(2A) adenosine receptor agonists ATL193 and ATL146e administered by either intravenous infusion or bolus injection. Circulation 104: 1181–1187, 2001.[Abstract/Free Full Text]
  24. Hannan RL, John MC, Kouretas PC, Hack BD, Matherne GP, Laubach VE. Deletion of endothelial nitric oxide synthase exacerbates myocardial stunning in an isolated mouse heart model. J Surg Res 93: 127–132, 2000.[CrossRef][Web of Science][Medline]
  25. Hein TW, Kuo L. cAMP-independent dilation of coronary arterioles to adenosine: role of nitric oxide, G proteins, and K(ATP) channels. Circ Res 85: 634–642, 1999.[Abstract/Free Full Text]
  26. Herskowitz A, Choi S, Ansari AA, Wesselingh S. Cytokine mRNA expression in postischemic/reperfused myocardium. Am J Pathol 146: 419–428, 1995.[Abstract]
  27. Heymann MA, Payne BD, Hoffman JI, Rudolph AM. Blood flow measurements with radionuclide-labeled particles. Prog Cardiovasc Dis 20: 55–79, 1977.[CrossRef][Web of Science][Medline]
  28. Heyndrickx GR, Millard RW, McRitchie RJ, Maroko PR, Vatner SF. Regional myocardial functional and electrophysiological alterations after brief coronary artery occlusion in conscious dogs. J Clin Invest 56: 978–985, 1975.[Web of Science][Medline]
  29. Homans DC, Laxson DD, Sublett E, Lindstrom P, Bache RJ. Cumulative deterioration of myocardial function after repeated episodes of exercise-induced ischemia. Am J Physiol Heart Circ Physiol 256: H1462–H1471, 1989.[Abstract/Free Full Text]
  30. Homans DC, Sublett E, Dai XZ, Bache RJ. Persistence of regional left ventricular dysfunction after exercise-induced myocardial ischemia. J Clin Invest 77: 66–73, 1986.[Web of Science][Medline]
  31. Jordan JE, Zhao ZQ, Sato H, Taft S, Vinten-Johansen J. Adenosine A2 receptor activation attenuates reperfusion injury by inhibiting neutrophil accumulation, superoxide generation and coronary endothelial adherence. J Pharmacol Exp Ther 280: 301–309, 1997.[Abstract/Free Full Text]
  32. Kaelin-Lang A, Lauterburg T, Burgunder JM. Expression of adenosine A2a receptor gene in rat dorsal root and autonomic ganglia. Neurosci Lett 246: 21–24, 1998.[CrossRef][Web of Science][Medline]
  33. Kim SJ, Peppas A, Hong SK, Yang G, Huang Y, Diaz G, Sadoshima J, Vatner DE, Vatner SF. Persistent stunning induces myocardial hibernation and protection: flow/function and metabolic mechanisms. Circ Res 92: 1233–1239, 2003.[Abstract/Free Full Text]
  34. Kloner RA, Allen J, Cox TA, Zheng Y, Ruiz CE. Stunned left ventricular myocardium after exercise treadmill testing in coronary artery disease. Am J Cardiol 68: 329–334, 1991.[CrossRef][Web of Science][Medline]
  35. Kloner RA, Jennings RB. Consequences of brief ischemia: stunning, preconditioning, and their clinical implications: part 2. Circulation 104: 3158–3167, 2001.[Abstract/Free Full Text]
  36. Lasley RD, Jahania MS, Mentzer RM Jr. Beneficial effects of adenosine A2a agonist CGS-21680 in infarcted and stunned porcine myocardium. Am J Physiol Heart Circ Physiol 280: H1660–H1666, 2001.[Abstract/Free Full Text]
  37. Lasley RD, Zhou Z, Hegge JO, Bunger R, Mentzer RM Jr. Adenosine attenuates in vivo myocardial stunning with minimal effects on cardiac energetics. Basic Res Cardiol 93: 303–312, 1998.[CrossRef][Web of Science][Medline]
  38. Li J, Fenton RA, Wheeler HB, Powell CC, Peyton BD, Cutler BS, Dobson JG Jr. Adenosine A2a receptors increase arterial endothelial cell nitric oxide. J Surg Res 80: 357–364, 1998.[CrossRef][Web of Science][Medline]
  39. Maddock HL, Broadley KJ, Bril A, Khandoudi N. Role of endothelium in ischaemia-induced myocardial dysfunction of isolated working hearts: cardioprotection by activation of adenosine A(2A) receptors. J Auton Pharmacol 21: 263–271, 2001.[CrossRef][Web of Science][Medline]
  40. McFalls EO, Baldwin D, Marx D, Jaimes D, Fashingbauer P, Ward HB. Adenosine receptor blockade enhances myocardial stunning without a sustained effect on fluorine-18-FDG uptake postreperfusion. J Nucl Med 39: 944–949, 1998.[Abstract/Free Full Text]
  41. McPherson JA, Barringhaus KG, Bishop GG, Sanders JM, Rieger JM, Hesselbacher SE, Gimple LW, Powers ER, Macdonald T, Sullivan G, Linden J, Sarembock IJ. Adenosine A(2A) receptor stimulation reduces inflammation and neointimal growth in a murine carotid ligation model. Arterioscler Thromb Vasc Biol 21: 791–796, 2001.[Abstract/Free Full Text]
  42. Meldrum DR. Tumor necrosis factor in the heart. Am J Physiol Regul Integr Comp Physiol 274: R577–R595, 1998.[Abstract/Free Full Text]
  43. Nicklas JM, Becker LC, Bulkley BH. Effects of repeated brief coronary occlusions on regional left ventricular function and dimension in dogs. Am J Cardiol 56: 473–478, 1985.[CrossRef][Web of Science][Medline]
  44. Olanrewaju HA, Mustafa SJ. Adenosine A(2A) and A(2B) receptors mediated nitric oxide production in coronary artery endothelial cells. Gen Pharmacol 35: 171–177, 2000.[Medline]
  45. Rieger JM, Brown ML, Sullivan GW, Linden J, Macdonald TL. Design, synthesis, and evaluation of novel A2A adenosine receptor agonists. J Med Chem 44: 531–539, 2001.[CrossRef][Web of Science][Medline]
  46. Saavedra WF, Paolocci N, St John ME, Skaf MW, Stewart GC, Xie JS, Harrison RW, Zeichner J, Mudrick D, Marban E, Kass DA, Hare JM. Imbalance between xanthine oxidase and nitric oxide synthase signaling pathways underlies mechanoenergetic uncoupling in the failing heart. Circ Res 90: 297–304, 2002.[Abstract/Free Full Text]
  47. Schwarz ER, Schaper J, vom Dahl J, Altehoefer C, Grohmann B, Schoendube F, Sheehan FH, Uebis R, Buell U, Messmer BJ, Schaper W, Hanrath P. Myocyte degeneration and cell death in hibernating human myocardium. J Am Coll Cardiol 27: 1577–1585, 1996.[Abstract]
  48. Schwarz ER, Schoendube FA, Kostin S, Schmiedtke N, Schulz G, Buell U, Messmer BJ, Morrison J, Hanrath P, vom Dahl J. Prolonged myocardial hibernation exacerbates cardiomyocyte degeneration and impairs recovery of function after revascularization. J Am Coll Cardiol 31: 1018–1026, 1998.[Abstract/Free Full Text]
  49. Sebastiao AM, Ribeiro JA. Adenosine A2 receptor-mediated excitatory actions on the nervous system. Prog Neurobiol 48: 167–189, 1996.[CrossRef][Web of Science][Medline]
  50. Sekili S, Jeroudi MO, Tang XL, Zughaib M, Sun JZ, Bolli R. Effect of adenosine on myocardial ‘stunning’ in the dog. Circ Res 76: 82–94, 1995.[Abstract/Free Full Text]
  51. Sekili S, McCay PB, Li XY, Zughaib M, Sun JZ, Tang L, Thornby JI, Bolli R. Direct evidence that the hydroxyl radical plays a pathogenetic role in myocardial "stunning" in the conscious dog and demonstration that stunning can be markedly attenuated without subsequent adverse effects. Circ Res 73: 705–723, 1993.[Abstract/Free Full Text]
  52. Shen YT, Vatner SF. Mechanism of impaired myocardial function during progressive coronary stenosis in conscious pigs. Hibernation versus stunning? Circ Res 76: 479–488, 1995.[Abstract/Free Full Text]
  53. Singh M, Saini HK. Resident cardiac mast cells and ischemia-reperfusion injury. J Cardiovasc Pharmacol Ther 8: 135–148, 2003.[Abstract/Free Full Text]
  54. Stahl Aversano TR, Becker LC. Selective enhancement of function of stunned myocardium by increased flow. Circulation 74: 843–851, 1986.[Abstract/Free Full Text]
  55. Stangl V, Baumann G, Stangl K, Felix SB. Negative inotropic mediators released from the heart after myocardial ischaemia-reperfusion. Cardiovasc Res 53: 12–30, 2002.[Abstract/Free Full Text]
  56. Sullivan GW, Rieger JM, Scheld WM, Macdonald TL, Linden J. Cyclic AMP-dependent inhibition of human neutrophil oxidative activity by substituted 2-propynylcyclohexyl adenosine A(2A) receptor agonists. Br J Pharmacol 132: 1017–1026, 2001.[CrossRef][Web of Science][Medline]
  57. Triana JF, Li XY, Jamaluddin U, Thornby JI, Bolli R. Postischemic myocardial "stunning". Identification of major differences between the open-chest and the conscious dog and evaluation of the oxygen radical hypothesis in the conscious dog. Circ Res 69: 731–747, 1991.[Abstract/Free Full Text]
  58. Yao Z, Gross GJ. Glibenclamide antagonizes adenosine A1 receptor-mediated cardioprotection in stunned canine myocardium. Circulation 88: 235–244, 1993.[Abstract/Free Full Text]
  59. Zhao M, Zhang H, Robinson TF, Factor SM, Sonnenblick EH, Eng C. Profound structural alterations of the extracellular collagen matrix in postischemic dysfunction ("stunned") but viable myocardium. J Am Coll Cardiol 10: 1322–1334, 1987.[Abstract]
  60. Zhu WX, Myers ML, Hartley CJ, Roberts R, Bolli R. Validation of a single crystal for measurement of transmural and epicardial thickening. Am J Physiol Heart Circ Physiol 251: H1045–H1055, 1986.[Abstract/Free Full Text]



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