Effect of Adenosine on Myocardial ‘Stunning’ in the Dog
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Abstract
Abstract Recent evidence suggests a cardioprotective effect of adenosine in myocardial ischemia and reperfusion. The present study was undertaken to determine (1) whether adenosine attenuates myocardial stunning, (2) if so, whether the beneficial effect of adenosine takes place during ischemia or after reperfusion, and (3) whether adenosine preconditions against myocardial stunning. A total of 93 dogs were used. In phase A of the study, open-chest dogs undergoing a 15-minute occlusion of the left anterior descending coronary artery followed by 4 hours of reperfusion received an intracoronary infusion of either saline (group I [control], n=14), 2 mg/min adenosine from 30 minutes before occlusion until 1 hour after reperfusion (group II, n=10), or 2 mg/min adenosine from 2 minutes before reperfusion until 1 hour after reperfusion (group III, n=11). Regional myocardial function (assessed as systolic wall thickening) was similar in the three groups at baseline and during ischemia. After reperfusion, dogs treated with adenosine before, during, and after ischemia (group II) demonstrated a significant improvement in the recovery of function that persisted throughout the 4 hours of reperfusion. In contrast, in dogs treated only during the reperfusion period (group III), the recovery of function was not statistically different from that in control dogs. The enhanced recovery effected by adenosine in group II could not be ascribed to differences in ischemic zone size, collateral flow during occlusion, coronary flow after reperfusion, arterial pressure, heart rate, or other hemodynamic variables. In phase B of the study, dogs received an intracoronary infusion of either saline (group IV [control], n=6) or adenosine (4 mg/min from 40 to 10 minutes before occlusion [group V, n=6]). Despite pretreatment with adenosine, the recovery of function in group V was indistinguishable from that in the control group. This study demonstrates that (1) continuous administration of adenosine before, during, and after ischemia results in a significant and sustained attenuation of myocardial stunning; (2) this improved recovery of function cannot be attributed to nonspecific variables, such as collateral flow during coronary occlusion, coronary flow after reperfusion, or other hemodynamic factors, and therefore reflects a direct cardioprotective action of adenosine; (3) the protection against stunning is lost or markedly diminished if adenosine is given only at reperfusion; and (4) administration of adenosine before ischemia does not precondition the myocardium against the stunning induced by a 15-minute occlusion. The failure of adenosine to produce a beneficial effect when given only at reperfusion indicates that in the 15-minute occlusion model, the nucleoside acts primarily by decreasing the severity of ischemic injury rather than by mitigating reperfusion injury.
Although numerous studies1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 have suggested a protective effect of adenosine in various settings of myocardial ischemia and reperfusion, most of these investigations have not specifically addressed the issue of reversible postischemic dysfunction or myocardial “stunning.”16 Indeed, only few experiments12 14 15 have examined the effect of adenosine in a model of “pure” myocardial stunning in vivo. Two of these studies12 15 investigated the role of endogenous adenosine by using an adenosine deaminase inhibitor with or without a nucleoside transport blocker and reported a beneficial effect. The other study14 found a salutary effect of an A1-receptor agonist in a model of multiple 5-minute ischemia/10-minute reperfusion cycles. To our knowledge, there are no reports in the literature addressing the issue of whether exogenous adenosine protects against myocardial stunning after a single brief (<20-minute) coronary occlusion in intact animals.
The beneficial effects previously observed with augmentation of endogenous adenosine12 15 do not necessarily imply that exogenous adenosine will also be protective. This is because (1) the concentration of adenosine achieved in the interstitium and myocytes with adenosine deaminase inhibitors and nucleoside transport blockers may be much higher than that which can possibly be achieved with exogenous adenosine17 (in fact, Berne18 recently demonstrated directly that only a small amount of exogenous adenosine reaches the interstitial compartment); (2) it is not known whether the beneficial effects of inhibiting adenosine catabolism and transport are due to decreased formation of free radicals via the xanthine oxidase reaction or to increased concentration of adenosine3 8 ; and (3) in contrast to adenosine deaminase inhibitors and nucleoside transport blockers, exogenous adenosine would be expected to increase oxyradical production via xanthine oxidase,17 an effect that might offset other beneficial actions. Thus, there are important differences between endogenous versus exogenous provision of adenosine, such that the effects of one mode of therapy may not apply to the other.
Furthermore, although it is well established that adenosine A1-receptor activation preconditions the heart against infarction and that the magnitude of this protection is dramatic,11 13 19 it is controversial whether A1-receptor activation can also precondition against stunning. Studies using multiple 5-minute coronary occlusions interspersed with 10 minutes of reperfusion have yielded conflicting results, with one study20 suggesting that adenosine receptor blockade prevents the preconditioning effect of the first occlusion and another14 suggesting that it does not. Studies using brief ischemic episodes followed by a longer ischemic episode have also yielded conflicting results. A recent report21 concluded that a 1-minute coronary occlusion attenuated the stunning induced by a subsequent 10-minute occlusion and that this effect was abolished by adenosine receptor blockade. In contrast, other studies22 23 found that brief (2- to 5-minute) ischemia did not precondition against the stunning induced by a 15-minute coronary occlusion. However, since the preconditioning protocols known to be most effective against infarction (ie, a 5-minute occlusion) induced stunning in themselves,22 23 the possibility remains that a preconditioning effect of these protocols did not become manifest because there was a cumulative effect between the dysfunction induced by the preconditioning 5-minute ischemia and the dysfunction induced by the 15-minute occlusion. A 2.5-minute and a 2-minute occlusion also failed to precondition against stunning22 23 ; although these short occlusions did not induce overt depression of contractility,22 23 it is nevertheless possible that they may have “sensitized” the myocardium to the dysfunction caused by the subsequent 15-minute occlusion.24 In an effort to overcome these problems, in the present investigation we substituted adenosine for ischemia as a preconditioning protocol, so that any protective effect of A1-receptor stimulation could be evaluated without impairing baseline contractile function.
The present study was undertaken to address three fundamental questions. First, what effect, if any, does exogenous adenosine have on myocardial stunning in intact animals? Second, if a beneficial effect does exist, is it due to attenuation of cellular perturbations that occur during ischemia (ischemic injury) or cellular perturbations that occur after reperfusion (“reperfusion injury”)? This is an important issue in view of the numerous reports2 4 6 10 indicating a reduction in infarct size when adenosine was administered at the time of reperfusion. Finally, does adenosine precondition against myocardial stunning?
Materials and Methods
A total of 93 dogs were used. Since the experimental preparation and techniques have been previously described in detail,15 25 26 27 28 29 30 31 32 they will not be detailed here.
Experimental Preparation and General Protocol
Pentobarbital-anesthetized dogs of either sex (13 to 34 kg) were instrumented with a snare and a Doppler flow probe around the mid left anterior descending coronary artery (LAD), a 27-gauge intracoronary needle just distal to the snare occluder, Doppler wall thickening probes, catheters in the aorta and left atrium, and a high-fidelity Millar pressure transducer in the left ventricular (LV) cavity. To prevent clotting, heparin was given immediately after insertion of the needle (3000 U IV) and thereafter (500 U/h). All dogs underwent a 15-minute LAD occlusion followed by 4 hours of reperfusion. Regional myocardial blood flow was determined by injecting radioactive microspheres, as previously described,25 before coronary occlusion (baseline), 8 to 10 minutes after occlusion, 25 minutes after reperfusion (during adenosine infusion), and 4 hours after reperfusion. Systolic thickening fraction was assessed by using the pulsed Doppler epicardial probe.15 25 26 27 28 29 30 31 32 The total deficit of wall thickening after reperfusion was calculated by measuring the area between the wall thickening–time line and the baseline (100% line) during the first 4 hours of reflow.30 31 The size of the occluded coronary vascular bed was determined by a postmortem dual-perfusion technique.15
Adenosine Treatment
In phase A, dogs were assigned to one of three intracoronary treatments. Group I (control group) received vehicle (heparinized normal saline) at a rate of 2 mL/min beginning 30 minutes before occlusion and ending 60 minutes after reperfusion, whereas groups II and III received adenosine. Adenosine (Sigma Chemical Co) was dissolved in heparinized saline at a final concentration of 1 mg/mL; the pH of the solution (which was ≈2.8) was adjusted to 7.4 by using 0.1 mol/L NaOH. In group II, the infusion of adenosine was started 30 minutes before coronary occlusion and ended 60 minutes after reperfusion. In group III, the infusion of adenosine was started 2 minutes before reperfusion and ended 60 minutes after reperfusion. In both groups, adenosine was infused at a rate of 2 mL/min, corresponding to a dose of 2 mg/min. In pilot studies, this was found to be the highest dose that could be given intracoronarily without producing hemodynamic effects (ie, arterial hypotension). During the first 13 minutes of LAD occlusion, the rate of infusion was reduced to 10% of preocclusion values (0.2 mL/min) in all three groups to avoid washout of catabolites and in group II to prevent the local concentration of adenosine in the ischemic tissue from achieving very high levels.
In phase B, dogs were assigned to one of two intracoronary treatments: (1) group IV (control group) received vehicle for a duration of 30 minutes at a rate of 2 mL/min beginning 40 minutes before coronary occlusion and ending 10 minutes before occlusion, and (2) group V received a 4-mg/min infusion of adenosine for a duration of 30 minutes starting 40 minutes before coronary occlusion and ending 10 minutes before occlusion. Adenosine was dissolved as in phase A, except that the final concentration was 2 mg/mL.
Statistical Analysis
Data are reported as mean±SEM. Continuous variables were analyzed by a two-way repeated-measures ANOVA to determine whether there was a main effect of group, a main effect of time, or a time by group interaction. If the global tests showed a significant main effect or interaction, post hoc contrasts between groups at various time points or between time points within one group were performed with Student’s t tests for unpaired or paired data, as appropriate, and the resulting P values were adjusted according to the Bonferroni correction.33 All analyses were performed using the SAS software system.34 Two-way ANOVA was performed by using the SAS procedure glm.34 The correlation between thickening fraction at 1 and 4 hours of reperfusion and transmural collateral blood flow was examined by using linear regression analysis, and comparisons between groups were made by ANCOVA, in which collateral flow was the covariate.
Results
Phase A: Effect of Adenosine on Myocardial Stunning
Of the 72 dogs initially anesthetized in phase A, 37 (51%) were excluded for the reasons outlined in Table 1⇓. The final analysis included 14 dogs in group I (control), 10 dogs in group II, and 11 dogs in group III. Postmortem triphenyltetrazolium chloride (TTC) staining confirmed the absence of infarction in all animals. Among the dogs that underwent coronary occlusion, the incidence of ventricular fibrillation tended to be higher in group II (6 [38%] of 16) than in group I (3 [18%] of 17) and group III (2 [15%] of 13), but the differences were not statistically significant (P=.37 versus group I and P=.36 versus group III [χ2 test with continuity correction]). It is important to point out that among the dogs that survived, there was no evidence that the severity of ischemia was less in group II than in groups I and III, since collateral flow and occluded bed size were similar in all three groups and the rate-pressure product during occlusion was actually higher in group II (see below).
Reasons for Excluding Dogs From the Study
Arterial Blood Gases, Hematocrit, and Temperature
As illustrated in Tables 2⇓ and 3⇓, the arterial pH, Po2, hematocrit, and the esophageal temperature were within physiological limits in all three groups throughout the experimental protocol (the hematocrit tended to decrease at 2 hours of reperfusion in group II but was not significantly different from baseline values).
Arterial Blood Gases and Hematocrit
Esophageal Temperature
Hemodynamic Variables
Table 4⇓ shows hemodynamic variables for phase A. The intracoronary administration of adenosine at a dose of 2 mg/min did not produce any substantial hemodynamic alteration other than a fourfold to fivefold increase in LAD blood flow. In groups II and III, LAD flow remained elevated until 1 hour after reperfusion (ie, until the end of adenosine infusion); however, by 2 hours of reperfusion, LAD flow had returned to baseline levels in both groups. Peak flow after reperfusion was similar in groups I, II, and III (76.7±6.7, 77.2±11.3, and 89.7±11.9 mL/min, respectively), and minimal vascular resistance was also similar (data not shown), indicating that adenosine did not increase the hyperemic response after reflow. Throughout the protocol, all measured variables (heart rate, systolic arterial pressure, rate-pressure product, left atrial pressure, LAD blood flow, and peak positive and negative LV dP/dt) were similar in the control and treated groups, with few exceptions. Compared with the control group (group I), group II exhibited a greater heart rate at baseline and a greater rate-pressure product at baseline and during coronary occlusion, two differences that, if anything, would be expected to exacerbate the severity of myocardial stunning. Peak positive LV dP/dt at 4 hours of reperfusion was greater in groups II and III than in the control group. However, none of these differences could account for the enhanced recovery of wall thickening observed in group II at 2 and 3 hours of reperfusion.
Hemodynamic Variables in Phase A
Occluded Bed Size
The size of the occluded vascular bed in the three groups was comparable, averaging 17.4±1.0 g (20.9± 1.1% of LV weight) in control dogs, 22.7±2.2 g (23.8 ±1.0%) in group II, and 18.1±1.8 g (20.9±1.7%) in group III.
Regional Myocardial Blood Flow
Table 5⇓ shows regional myocardial blood flow for phase A. At baseline, blood flow to both the ischemic and the nonischemic zone was significantly (P<.05) greater in groups II and III than in the control group, possibly because of the higher oxygen demands associated with the higher heart rate-pressure product (Table 4⇑). This difference persisted at 4 hours of reperfusion (P<.05); however, when blood flow to the postischemic region at 4 hours of reperfusion was expressed as a percentage of nonischemic zone flow, no difference was observed among the three groups, indicating that the higher flows noted in the stunned myocardium in groups II and III were a consequence of the baseline flow differences rather than the manifestation of a protective effect of adenosine on the postischemic vasculature. During coronary occlusion, the average blood flow to the ischemic region was similar in control and treated groups, both in absolute terms (milliliters per minute per gram) and as a percentage of nonischemic zone flow. As expected, at 25 minutes after reperfusion (during adenosine infusion), mean transmural blood flow to the previously ischemic region was markedly elevated in both groups II and III (3.7 and 5.6 times baseline flow, respectively).
Regional Myocardial Blood Flow in Phase A
Regional Myocardial Function
The systolic thickening fraction in the nonischemic (control) region did not differ significantly among the three groups at any time point during the protocol (Table 4⇑). Baseline systolic thickening fraction in the region to be rendered ischemic was 23.7±2.0%, 25.5±2.0%, and 28.3±2.1% in groups I, II, and III, respectively (P=NS). In group II, the thickening fraction decreased significantly during infusion of adenosine (to 84.1±3.6% of baseline, P<.01), indicating a negative inotropic effect of the nucleoside. During coronary occlusion, the extent of paradoxical systolic thinning in the ischemic region was similar in all three groups (Fig 1⇓). After reperfusion, control dogs (group I) exhibited little recovery of contractile function, and 4 hours after restoration of flow, the previously ischemic region was still dyskinetic, indicating severe myocardial stunning. Two-way ANOVA demonstrated that after reperfusion there was a significant time-by-group interaction, indicating that the changes in thickening fraction over time were different in the three groups. As Fig 1⇓ illustrates, in group II the recovery of function was enhanced throughout the 4-hour observation period; post hoc Student’s t tests with the Bonferroni correction confirmed that the thickening fraction was significantly greater than in the control group at 30 minutes after reflow and at all subsequent time points. In contrast, in group III post hoc tests demonstrated that the postischemic recovery of function did not differ significantly from that in the control group (Fig 1⇓). In comparison with the control group, the total deficit of wall thickening during the entire 4-hour period of reperfusion (an integrative assessment of postischemic dysfunction30 31 was reduced by 38% in group II (P<.01) and by only 14% in group III (P=NS).
Graph showing systolic thickening fraction in the ischemic-reperfused region 20 minutes into the infusion of adenosine (ADO) (ie, 10 minutes before coronary occlusion), 5 minutes after coronary occlusion (O), and at selected times after reperfusion in phase A. Group I indicates control dogs; group II, dogs treated with adenosine continuously before, during, and after ischemia; and group III, dogs treated with adenosine beginning at reperfusion. Thickening fraction is expressed as a percentage of baseline values. Data are mean±SEM.
As shown in Fig 2⇓, in control dogs wall thickening at 1 and 4 hours after reperfusion was directly related to collateral flow during coronary occlusion (r=.59 and r=.53, respectively). ANCOVA demonstrated that in group II this relation was altered, so that for the same level of collateral flow, thickening fraction after reperfusion was greater than in the control group by an average of 43% at 1 hour (P<.01) and 69% at 4 hours (P<.01) after reperfusion. ANCOVA demonstrated that the wall thickening–collateral flow relation was also significantly (P<.05) different between group II and group III. These results indicate that the enhanced recovery of wall thickening effected by adenosine infusion in group II was independent of any differences in blood flow during ischemia.
Scatterplots showing the relation between mean transmural collateral blood flow to the ischemic region during coronary occlusion (horizontal axis) and systolic wall thickening (vertical axis) at 1 and 4 hours after reperfusion in phase A. Group I indicates control dogs (n=14); group II, dogs treated with adenosine continuously before, during, and after ischemia (n=10); and group III, dogs treated with adenosine beginning at reperfusion (n=11). Collateral flow is expressed in milliliters per minute per gram, and thickening fraction is expressed as a percentage of baseline values (collateral flow is not expressed as a percentage of nonischemic zone flow, because nonischemic zone flow during coronary occlusion may have been altered by adenosine). Note that at any given collateral flow, group II exhibited greater wall thickening than did group I or group III. The linear regression equations for group I were as follows: 1 hour, y=−71.5+245.4x (r=.59, P<.05); 4 hours, y=−68.7+278.0x (r=.53, P=.05). The equations for group II were as follows: 1 hour, y=−11.8+143.8x (r=.61, P=.06); 4 hours, y=33.7+77.7x (r=.62, P=.06). The equations for group III were as follows: 1 hour, y=−59.0+304.7x (r=.78, P<.01); 4 hours, y=−45.6+233.1x (r=.45). ANCOVA demonstrated that the two regression lines for groups I and II were significantly different (P<.01) and that at comparable flows, thickening fraction in group II exceeded that in control dogs by an average of 43% at 1 hour and 69% at 4 hours. Furthermore, ANCOVA demonstrated that the regression lines for groups II and III were also different (P<.05).
To determine whether adenosine-induced hyperemia increases systolic wall thickening in the stunned myocardium (and thus may account for the increased wall thickening observed in group II at 30 minutes and 1 hour), at 4 hours of reperfusion adenosine was infused intracoronarily for 10 minutes at a rate of 2 mg/min, which did not produce any systemic hemodynamic effects, in nine dogs from groups I or III (Fig 3⇓). Despite an average 4.4-fold increase in LAD flow, the thickening fraction did not change significantly (−29.8±11.7% of baseline before adenosine versus −14.7±12.0% during adenosine, P=.14 [Fig 3⇓]).
Graph showing systolic thickening fraction in the ischemic-reperfused region 4 hours after reperfusion and during infusion of adenosine (ADO) (ie, 10 minutes later) in nine dogs from either group I or group III. LAD indicates left anterior descending coronary artery. Thickening fraction is expressed as a percentage of baseline values. Solid dots indicate mean±SEM.
Power Analysis
A statistical analysis was performed to measure the probability that an improvement of wall thickening by adenosine given at reperfusion (group III) was missed because of insufficient sample size (type II error). Since in group II adenosine infused before, during, and after ischemia increased wall thickening at 4 hours of reperfusion by an average of 72% of baseline values compared with the control group (group I) and decreased the total postischemic deficit of wall thickening during the entire 4-hour period of reperfusion by an average of 38% compared with the control group, we measured the power of our experiment to demonstrate similar effects in group III. All power calculations were made at α=.05. The calculations revealed that there was a 94% probability of demonstrating a ≥72% (of baseline values) improvement of wall thickening in group III dogs versus control dogs at 4 hours of reperfusion, if this was the true effect of adenosine in group III. The chance of demonstrating a ≥38% reduction in the total deficit of wall thickening in group III dogs compared with control dogs was 84%.
On the other hand, the demonstration of smaller beneficial effects of adenosine given at reperfusion would have been difficult. For example, if the true increase in wall thickening effected by adenosine given at reperfusion was the same as that observed in our study in group III (ie, an increase of 15% of baseline values over the control group) and assuming that the variance of the samples was the same as that observed in our study, >120 dogs would have had to be studied in both groups I and III (total of >240 dogs) for this difference to be significant at P<.05. If the true decrease in the total postischemic deficit of wall thickening effected by adenosine given at reperfusion was the same as that observed in our study in group III (ie, a 14% decrease versus the control group) and assuming that the variance of the samples was the same as that observed in our experiment, 72 dogs would have had to be studied in both groups III and I (total of 144 dogs) for this difference to be statistically significant. In conclusion, if adenosine given at reperfusion exerted beneficial effects comparable to those produced by adenosine given before, during, and after ischemia, it is unlikely that such effects would have remained undetected in the present experiment; detection of smaller effects would have required prohibitively large sample sizes.
Phase B: Effect of Adenosine on Preconditioning Against Myocardial Stunning
Of the 21 dogs initially anesthetized in phase B, 9 (43%) were excluded for the reasons outlined in Table 1⇑. Thus, the final analysis included 6 control dogs (group IV) and 6 dogs pretreated with adenosine (group V). As in phase A, postmortem TTC staining confirmed the absence of infarction in all animals.
Arterial Blood Gases, Hematocrit, and Temperature
As shown in Tables 2⇑ and 3⇑, the arterial pH, Po2, hematocrit, and the esophageal temperature were within physiological limits in both groups.
Hemodynamic Variables
Table 6⇓ shows hemodynamic variables for phase B. In group V, the intracoronary administration of adenosine at a dose of 4 mg/min resulted in a 3.7-fold increase in LAD blood flow (as measured by Doppler flow probe) and in a 15-mm Hg drop in diastolic arterial pressure (95±8 mm Hg at baseline versus 80±10 mm Hg during adenosine, P<.05). Both the LAD flow and the diastolic pressure returned to baseline values during the 10-minute interval between adenosine administration and coronary occlusion. Throughout the protocol, all measured variables (heart rate, systolic arterial pressure, rate-pressure product, left atrial pressure, LAD blood flow, and peak positive and negative LV dP/dt) were similar in the control and treated groups, with the exception of left atrial pressure, which was greater in group V than in the control group at baseline, before occlusion, and at several time points after reperfusion.
Hemodynamic Variables in Phase B
Occluded Bed Size
The size of the occluded vascular bed did not differ in the two groups (16.6±1.1 g [22.9±1.9% of LV weight] in the control group and 19.8±2.6 g [20.2±1.9%] in group V).
Regional Myocardial Blood Flow
Table 7⇓ shows regional myocardial blood flow for phase B. There were no significant differences between the two groups with respect to regional myocardial blood flow at baseline, during LAD occlusion, and 4 hours after reperfusion. In group V, the mean transmural blood flow to the soon-to-be-ischemic zone increased 4.2-fold during infusion of adenosine. At the same time, flow to the nonischemic zone increased 2.4-fold (P<.05), as a result of recirculation of adenosine. In both groups, flow to the postischemic subendocardium at 4 hours of reperfusion was significantly decreased compared with baseline values (P<.01 in group IV and P<.05 in group V).
Regional Myocardial Blood Flow in Phase B
Regional Myocardial Function
Systolic thickening fraction in the nonischemic (control) region did not differ significantly between the two groups (Table 6⇑). Baseline systolic thickening fraction in the region to be rendered ischemic was 23.6±2.8% in the control group and 24.5±2.1% in group V (P=NS). In group V, thickening fraction decreased transiently during the infusion of adenosine (81.4±4.8% of baseline values, P<.01) (Fig 4⇓), in analogy with the results obtained in group II. After discontinuation of adenosine (immediately before coronary occlusion), however, thickening fraction returned to values not significantly different from baseline (91.2±4.5% of baseline). During coronary occlusion, the degree of paradoxical systolic wall thinning was comparable in groups IV and V (Fig 4⇓). After reperfusion, there was no appreciable difference in the recovery of contractile function between the two groups, both of which exhibited severe stunning (Fig 4⇓). Two-way ANOVA failed to show a significant main effect for group or a group by time interaction. Fig 5⇓ illustrates the relation between postischemic wall thickening and collateral flow for these two groups. Note that there is overlap between groups IV and V at all levels of collateral flow. ANCOVA failed to reveal any difference between the two groups.
Graph showing systolic thickening fraction in the ischemic-reperfused region 20 minutes into the infusion of adenosine (ADO) (ie, 20 minutes before coronary occlusion), 1 minute before coronary occlusion (Pre-O) (ie, 9 minutes after the discontinuation of adenosine infusion), 5 minutes after coronary occlusion (O), and at selected times after reperfusion in phase B. Group IV indicates control dogs; group V, dogs treated with adenosine before ischemia. Thickening fraction is expressed as a percentage of baseline values. Data are mean±SEM.
Scatterplots showing the relation between mean transmural collateral blood flow to the ischemic region during coronary occlusion (horizontal axis) and systolic wall thickening (vertical axis) at 1 and 4 hours after reperfusion in phase B. Group IV indicates control dogs (n=6); group V, dogs treated with adenosine before ischemia (n=6). Collateral flow is expressed in milliliters per minute per gram, and thickening fraction is expressed as a percentage of baseline values (collateral flow is expressed in milliliters per minute per gram for consistency with Fig 2⇑). Note that there is considerable overlap between the two groups at all levels of collateral flow. The linear regression equations for group IV were as follows: 1 hour, y=−34.4+ 15.0x (r=.05, P=NS); 4 hours, y=−45.4+74.6x (r=.43, P=NS). The equations for group V were as follows: 1 hour, y=−45.2+102.8x (r=.41, P=NS); 4 hours, y=−18.7−10.6x (r=.04, P=NS). ANCOVA demonstrated that there was no significant difference between the two groups.
Power Analysis
As in phase A, we measured the probability that a beneficial effect of adenosine pretreatment was missed in group V (type II error). Because infusion of adenosine in group II increased wall thickening at 4 hours of reperfusion by an average of 72% of baseline values compared with the control group, we measured the power of our experiment to demonstrate a similar effect in group V. Power calculations revealed that there was an 81% probability of demonstrating a ≥72% (of baseline values) improvement of wall thickening in group V compared with the control group at 4 hours of reperfusion, if this was the true effect of adenosine pretreatment. Thus, if pretreatment with adenosine produces an appreciable beneficial effect on myocardial stunning, it is unlikely that such an effect would have remained undetected in the present study. On the other hand, if the true increase in wall thickening effected by adenosine pretreatment was the same as that observed in group V (ie, only an increase of 13% of baseline values compared with the control group) and assuming that the variance of the samples was the same as that observed in our study, it would have been necessary to study >120 dogs in both groups IV and V (total of >240 dogs) for this difference to become significant at P<.05. Thus, the demonstration of minor beneficial effects of adenosine pretreatment would have required prohibitively large sample sizes.
Discussion
To our knowledge, this is the first study to demonstrate that (1) exogenous adenosine mitigates the severity of postischemic myocardial dysfunction after a single brief ischemic insult in the intact animal; (2) this improved recovery of function cannot be ascribed to nonspecific variables, such as collateral blood flow during coronary occlusion, ischemic zone size, coronary flow after reperfusion, or other hemodynamic factors, and therefore reflects a direct cardioprotective effect of adenosine; (3) the primary mechanism of the beneficial action of adenosine in myocardial stunning is a decrease in the severity of ischemic injury rather than reperfusion injury; and (4) administration of intracoronary adenosine does not precondition the myocardium against the stunning associated with a 15-minute coronary occlusion. Each of these points requires further discussion and will be addressed below.
Effect of Adenosine on Myocardial Stunning
When adenosine was given continuously before, during, and after ischemia (group II), a significant and sustained attenuation of stunning was observed, which persisted throughout 4 hours of reperfusion despite the fact that adenosine infusion was discontinued after 1 hour of reperfusion. Since LAD blood flow returned to baseline values within minutes after stopping the infusion of adenosine, the improved recovery of function observed at 2, 3, and 4 hours of reperfusion cannot be attributed to increased myocardial perfusion (ie, to a Gregg phenomenon35 ). Although at 30 minutes and 1 hour of reperfusion LAD flow was approximately fourfold higher in group II compared with group I (Table 4⇑), several considerations make it unlikely that this hyperemia accounted for the difference in wall thickening observed at these time points. First, in group II we found that when LAD flow returned to baseline values (≈10 minutes after adenosine was discontinued), the thickening fraction did not decrease significantly compared with the 1-hour values measured during hyperemia (Δ= −7.4±12.9%, P=NS). Second, if the enhanced wall thickening observed at 30 minutes and 1 hour in group II was due to the hyperemia, one would have expected a similar beneficial effect in group III, which exhibited a similar hyperemia at 30 minutes and 1 hour (Table 4⇑). Finally, infusion of adenosine at 4 hours failed to increase thickening fraction in the stunned myocardium despite a 4.4-fold increase in LAD flow (Fig 3⇑).
There were no other differences that could have accounted for the enhanced recovery in group II. This group was similar to groups I and III with respect to collateral flow, ischemic zone size, and most of the hemodynamic variables. For reasons that are unclear, group II had a higher heart rate and rate-pressure product at baseline and a higher rate-pressure product during occlusion (Table 4⇑), but these differences, if anything, should have exacerbated the severity of ischemia and thus should have led to an underestimation of the beneficial effects of adenosine. It should be noted that at 3 and 4 hours of reperfusion, the heart rate and rate-pressure product were similar in groups II and III (Table 4⇑), yet the recovery of function was significantly enhanced only in group II. Thus, we conclude that continuous infusion of adenosine before, during, and after ischemia reduces the severity of myocardial stunning by a mechanism that is independent of nonspecific factors, such as coronary flow or systemic hemodynamics.
Does Adenosine Attenuate Ischemic Injury or Reperfusion Injury?
In contrast to group II, group III exhibited no statistically significant improvement in the recovery of function when compared with the control group. The dose of adenosine used in group III (2 mg/min) was the same as that used in group II, and the fact that the infusion was started intracoronarily 2 minutes before the release of occlusion ensured that high local levels of adenosine were present in the LAD region at the very onset of reperfusion. Power analysis showed that there was a >90% probability that if adenosine given at reperfusion (group III) improved recovery of function to the same extent as adenosine given before, during, and after ischemia (group II), such an effect would have been detected in our study. With our sample sizes, we cannot rule out the possibility that adenosine given at reperfusion could exert a small protective effect (eg, an improvement of wall thickening of 15% of baseline values), but the physiological significance of such an effect would be questionable.
Thus, our results indicate that in the 15-minute occlusion model, administration of adenosine only during the reperfusion phase is either ineffective or much less effective than continuous administration of adenosine before ischemia, during ischemia, and during reperfusion. This implies that adenosine does not act primarily to decrease the component of “reperfusion injury” that contributes to the development of myocardial stunning.36 (Such a component may still be decreased indirectly, however, as a result of the ability of adenosine to attenuate ischemic injury, since the severity of reperfusion injury in stunned myocardium appears to be modulated by the severity of the antecedent ischemic injury.26 27 28 29 31 36 On the other hand, the results of phase B clearly demonstrate that administration of adenosine only before ischemia fails to enhance the postischemic recovery of function, implying that the beneficial effects observed in group II were not due to the 30-minute infusion of adenosine given before coronary occlusion in this group. Taken together, the results of phases A and B indicate that in the 15-minute coronary occlusion model, the attenuation of myocardial stunning by adenosine is due to a beneficial effect that takes place mainly during the period of ischemia, not before ischemia or after reperfusion; ie, the primary mechanism of action of adenosine appears to be a reduction in the severity of the cellular perturbations that occur during ischemia (ischemic injury).
Does Adenosine Precondition Against Stunning?
In phase B, we sought to determine whether adenosine can precondition against stunning. The impetus to test this idea was provided by the demonstration that activation of adenosine A1-receptors preconditions the myocardium against infarction11 13 19 and by recent reports20 21 suggesting that brief ischemia preconditions against stunning via an adenosine receptor–mediated mechanism. Our results clearly demonstrate that pretreatment with adenosine failed to protect the myocardium against subsequent stunning, despite the fact that adenosine was given for a relatively long time (30 minutes) and at a dose (4 mg/min) twofold greater than the dose (2 mg/min) found to be protective in group II. Thus, although adenosine preconditions against infarction,11 13 19 it does not precondition against the stunning induced by a 15-minute coronary occlusion. This finding further corroborates the concept that the pathogenesis and pathophysiology of myocardial stunning (a reversible form of injury) differ importantly from those of irreversible cellular injury (ie, myocardial infarction).36
We elected to use a higher infusion rate in group V than in group II (4 versus 2 mg/min, respectively) to maximize the chances of detecting a preconditioning effect, if one truly existed. Since exogenous adenosine is rapidly taken up by red blood cells and endothelial cells, we reasoned that a higher infusion rate would be more likely to produce sufficient myocardial A1-receptor activation to induce a preconditioning effect. The failure of the 4-mg/min infusion to precondition against stunning is unlikely to be due to toxicity because (1) the hemodynamic effects were very mild (only a transient decrease in diastolic arterial pressure with no change in systolic pressure or heart rate); (2) these effects resolved promptly after the infusion of adenosine was stopped, ie, before coronary occlusion; and (3) similar doses of adenosine (3.75 mg/min IC) have been shown to be protective in models of infarction.2 4
Previous Studies of Whether Adenosine Treatment Attenuates Myocardial Stunning
In a previous study,37 a bolus of adenosine given at the time of reperfusion was found to accelerate the rate of early contractile recovery (without affecting the final recovery) in stored or heterotopically transplanted rat hearts studied in vitro; this beneficial effect was associated with a sustained improvement in coronary flow. The difference between these findings and our present results may reflect differences in experimental preparations, species, and/or severity of ischemic injury.
Although many studies have suggested a beneficial effect of adenosine on postischemic myocardial dysfunction in a variety of experimental models,1 2 3 4 5 8 9 10 12 14 15 extrapolation of these results to the setting of regional myocardial stunning after reversible ischemia would be problematic. Many of these studies3 5 8 9 12 15 examined the effects of inhibitors of adenosine catabolism and transport, not the effect of exogenous adenosine. Some studies1 5 9 were conducted in isolated hearts, so that the results cannot necessarily be extrapolated to intact animals. Other studies2 4 10 involved models of prolonged (90- to 120-minute) ischemia resulting in subendocardial infarction, which differ from models of “pure” stunning caused by a brief (<20-minute), completely reversible ischemic insult.36 Since the amelioration of postischemic dysfunction noted in these studies2 4 10 was associated with a decrease in infarct size, one cannot comment on the specific effects on myocardial stunning. Finally, two studies3 8 used a model of prolonged global ischemia with cardioplegic arrest and cardiopulmonary bypass, which again differs importantly from brief, regional ischemia.36
To our knowledge, only three full-length reports12 14 15 have addressed the role of adenosine on myocardial stunning after reversible ischemia in vivo. Using an open-chest canine model of 15 minutes of coronary occlusion followed by 1 hour of reperfusion, Dorheim et al12 observed a modest attenuation of postischemic dysfunction in dogs treated with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an inhibitor of adenosine deaminase. Zughaib et al15 demonstrated that augmentation of endogenous adenosine with EHNA plus 6-(4-nitrobenzyl)mercaptopurine ribonucleoside (NBMPR), a nucleoside transport inhibitor, resulted in attenuation of myocardial stunning in dogs subjected to a 15-minute coronary occlusion followed by 4 hours of reperfusion. Unlike the present study, the purpose of these previous studies12 15 was to examine the effects of increasing the levels of endogenous adenosine by inhibiting its metabolism and transport. However, inhibitors of adenosine catabolism and transport may act not only by augmenting adenosine levels but also by preventing the generation of oxygen radicals from xanthine and hypoxanthine; therefore, it is not possible to discern whether the protective effects of EHNA and NBMPR in these studies12 15 were due to the former or to the latter mechanism. In open-chest dogs subjected to six 5-minute occlusion/10-minute reperfusion cycles followed by 2 hours of reperfusion, Yao and Gross14 found a significant beneficial effect when a selective adenosine A1-receptor agonist was infused continuously before and during the six cycles and a significant exacerbation of stunning when the animals were pretreated with an A1-receptor antagonist. Unlike the present investigation, their study14 examined a model of stunning induced by repetitive ischemia, which differs in several respects from models of stunning induced by a single coronary occlusion.36 Our results expand the findings of Dorheim et al,12 Zughaib et al,15 and Yao and Gross14 by demonstrating that (1) exogenous adenosine ameliorates myocardial stunning after a single ischemic insult, (2) this beneficial effect is sustained for 4 hours after reperfusion, (3) it requires the administration of adenosine during ischemia, and (4) adenosine does not precondition against the stunning induced by a 15-minute coronary occlusion. Since exogenous adenosine should, if anything, increase oxyradical production through the xanthine oxidase reaction, our results imply that the salutary effects observed with EHNA alone12 and EHNA+NBMPR15 were indeed mediated, at least in part, by augmentation of adenosine.
Previous Studies of Whether A1-Receptor Activation Preconditions Against Stunning
In a preliminary report in isolated working rat hearts, Cave et al38 found that a 5-minute period of perfusion with adenosine (10, 50, and 100 μmol/L) followed by a 5-minute adenosine-free perfusion period failed to increase the recovery of function after a subsequent 20-minute period of global ischemia. Although it is unclear whether the dysfunction observed in this model represents solely stunning, the results of Cave et al are in agreement with our conclusion that adenosine pretreatment does not mitigate myocardial stunning.
The preconditioning effects of adenosine A1-receptors against stunning in vivo are controversial. In a rabbit model of stunning induced by a 10-minute coronary occlusion, Urabe et al21 found that a prior 1-minute occlusion resulted in enhanced recovery of wall thickening and that the adenosine receptor antagonist 8-phenyltheophylline blocked this beneficial effect. However, other studies in dogs22 and pigs23 have failed to demonstrate a preconditioning effect of brief (2- to 5- minute) coronary occlusions against the stunning induced by a subsequent 15-minute occlusion. In a rabbit model of stunning induced by four cycles of 5-minute coronary occlusion/10-minute reperfusion, Bunch et al20 found that the adenosine receptor antagonist PD 115199 caused a progressive deterioration of function after each cycle, suggesting that the adenosine produced during the first ischemic episode preconditioned against the subsequent episodes. In contrast, using a canine model of stunning induced by six 5-minute occlusion/10-minute reperfusion cycles, Yao and Gross14 found that the adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine decreased the recovery of function after the first cycle but caused no additional deterioration after the next five cycles, suggesting that the adenosine released during the first ischemic episode was protective during that episode but did not precondition against the stunning induced by subsequent episodes.
The reason(s) for the apparent discrepancy among these previous studies14 20 21 22 23 is unclear but may relate to differences in experimental models and/or species. It should be noted that no previous study has examined the ability of adenosine (or adenosine agonists) to precondition against the stunning induced by a 15-minute coronary occlusion in the dog.
Mechanism of Adenosine-Mediated Protection
Adenosine exerts a number of actions that could beneficially affect myocardial stunning (reviewed in References 17, 39, and 4017 39 40 ). Theoretically, adenosine may reduce free radical formation by inhibiting lipolysis (and thus decreasing lipid peroxidation) and/or by decreasing auto-oxidation of catecholamines as a result of the inhibition of norepinephrine release.17 39 Adenosine may also protect against the effects of oxygen metabolites.41 However, our results clearly demonstrate that the primary mechanism of action of adenosine in this model of stunning is a decrease in the severity of ischemic injury, not reperfusion injury. Mitigation of ischemic injury by adenosine may take place via various mechanisms. For example, adenosine may attenuate calcium overload during ischemia by inhibiting calcium influx through the L-type calcium channel.17 40 42 Adenosine may also improve energy balance during ischemia by stimulating glycolysis,43 by inhibiting the release of norepinephrine from nerve endings,44 and/or by antagonizing the effect of catecholamines on contractility.39 45 46 In this regard, in the present study adenosine exerted a negative inotropic effect, as evidenced by a 15% to 20% decrease in wall thickening before coronary occlusion in both groups II and V (Figs 1⇑ and 4⇑), which is consistent with previous observations.47 48 49 50 51 Therefore, it is possible that the protection afforded by adenosine in group II was due in part to its antiadrenergic effects reflected by the decrease in ventricular contractility. Interestingly, adenosine failed to enhance recovery in group V, in which ischemia was initiated 10 minutes after discontinuation of the adenosine infusion, when the negative inotropic effect had largely dissipated (Fig 4⇑).
Our finding that adenosine acts primarily by mitigating ischemic injury is not in conflict with the notion that a free radical–mediated reperfusion injury plays a prominent role in the pathogenesis of myocardial stunning. First, it has been pointed out32 36 52 that free radicals are not the sole factor contributing to the pathogenesis of myocardial stunning. Second, and most important, since the magnitude of free radical generation after reperfusion in the stunned myocardium is directly related to the severity of the antecedent ischemic insult,26 27 28 29 31 36 any intervention that alleviates the injury incurred during ischemia would be expected to indirectly attenuate the radical-mediated injury inflicted after reperfusion.
Conclusions
In summary, the present study demonstrates that infusion of adenosine before, during, and after a 15-minute coronary occlusion results in a significant and sustained improvement in the recovery of myocardial function, which reflects a direct cardioprotective action of adenosine. This beneficial effect cannot be reproduced when adenosine is infused solely at reperfusion, indicating that in the 15-minute occlusion model, the purine acts primarily by decreasing the severity of ischemic injury. Intracoronary adenosine fails to precondition against myocardial stunning, a finding that emphasizes the differences between the pathophysiology of stunning and that of infarction. Although adenosine itself would not be a practical clinical therapy, adenosine receptor agonists or adenosine modulators may be useful in the management of myocardial stunning in patients.
Acknowledgments
This study was supported in part by National Institutes of Health (NIH) grant HL-43151 and SCOR grant HL-42267 (Dr Bolli) and by the Veterans Affairs Research Advisory Group Program (Dr Jeroudi). We thank Jennifer S. Pocius and Alejandro Tumang for excellent technical assistance and Valerie R. Price for expert secretarial assistance. Ultrasonic probes were provided by Dr Craig J. Hartley with the support of NIH grant HL-22512.
- Received April 12, 1994.
- Accepted October 3, 1994.
- © 1995 American Heart Association, Inc.
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- Effect of Adenosine on Myocardial ‘Stunning’ in the DogSelim Sekili, Mohamed O. Jeroudi, Xian-Liang Tang, Marcel Zughaib, Jian-Zhong Sun and Roberto BolliCirculation Research. 1995;76:82-94, originally published January 1, 1995https://doi.org/10.1161/01.RES.76.1.82
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