To date, only one published study has identified a protective role for the adenosine A₃ receptor subtype when activated at reperfusion using infarct size as the end point of injury in rat hearts (Maddock et al., 2002). The present study examined the effect of the adenosine A₃ receptor agonist, IB-MECA, on infarct size when added at reperfusion following a period of no-flow global ischaemia.

Coronary circulation of male Dunkin-Hartley guinea pig (300-350g) hearts was performed by a modification of the Langendorff method (Broadley, 1979). The perfusion fluid was delivered in a retrograde direction down the aorta at a constant flow rate of 7ml/min with the entire process from cervical dislocation to full flow of perfusate controlled to a time period of 2.5min. The Krebs bicarbonate solution was pre-warmed to 32°C and gassed with 5% CO₂ in oxygen before passing to the flow inducer, warming coil (37°C±0.5°C) and aortic cannula. Alterations in perfusion pressure were detected using a pressure transducer with a Condon mercury manometer included in series. A resting tension of 1g was applied and isometric tension recorded. No-flow global ischaemia was induced for 60min after 30min equilibration followed by 10min reperfusion at 30% then 60min of reperfusion at 100% of the pre-ischaemic flow rate. The selective adenosine A1 receptor antagonist, DPCPX (2´10^-7M), was added to the heart via constant perfusion 15min before ischaemia and during reperfusion. At the end of reperfusion, the ventricles were cut into 4 transverse slices before being incubated in 1% triphenyltetrazolium for 5-7min at 37°C±0.5°C and fixed in 10% formalin solution for 24hr. Non stained infarcted tissue was marked by an independent investigator and the area (sum of 4 sections) determined by computerized planimetery (SigmaScan Pro 5) and expressed as a percentage of the total heart area. Statistical comparisons were made using ANOVA followed by either a Student's unpaired t-test or the Bonferroni multiple comparisons test, P<0.05 indicating significance. There was no significant difference between the pre-ischaemic contractile tension, diastolic tension, heart rate and coronary perfusion pressure between any of the different experimental groups.

In contrast to the red brick stained viable myocardium, 38.8±5.1% (n=6) of the total heart area was infarct and remained unstained following 60min of no-flow global ischaemia. Hearts were randomly assigned to either the IB-MECA vehicle (0.01% DMSO) or drug treatment. IB-MECA (0.3µM, 4nM, 1nM) when added immediately at reperfusion via a slow infuser (0.25ml/min) had no significant effect on the percentage of infarct area (46.2±4.4%, n=7; 38.1±4.2%, n=6; 30.3±4.9%, n=7, respectively) compared to the vehicle (38.6±4.1%, n=11) (P>0.05). However, when DPCPX was excluded, IB-MECA (4nM) significantly reduced the percentage of infarct area (26.2±3.5%, n=7) compared to the vehicle (47.2±7.4%, n=6) (P<0.05). This cardioprotection was without a significant effect on the recovery of contractile tension after 10min of reperfusion at 100% of the pre-ischaemic flow rate (9.0±1.2% of 6.4±0.6g, n=7) compared to the vehicle (12.6±2.1% of 6.5±0.2g, n=6) (P>0.05). Diastolic tension increased during 30% (4.6±0.4g) and 100% reperfusion (2.0±0.3g, n=6) in vehicle treated controls. This contracture was not significantly different in IB-MECA (4nM) treated hearts (3.6±1.0g and 2.8±0.5g, n=7, respectively) (P>0.05).

The results suggest that the cardioprotective effect of IB-MECA against infarct size is due to a specific effect upon adenosine A₁ receptors and not mediated via activation of adenosine A₃ receptors.

Broadley, K.J. (1979). J. Pharmacol. Methods, 2, 143-156.
Maddock, H.L. et al., (2002). Am. J. Physiol., 283, H1307-H1313.

Supported by a British Heart Foundation studentship to L.Y.