Previous studies have shown that high-level (300-fold normal) cardiac overexpression of A1-adenosine receptors (A1-ARs) in transgenic (TG) mice protects isolated hearts against ischemiareperfusion injury. However, this high level of overexpression is associated with bradycardia and increased incidence of arrhythmia during ischemia in intact mice, which interfered with studies to determine whether this line of TG mice might also be protected against myocardial infarction (MI) in vivo. For these studies, we therefore selected a line of TG mice that overexpresses the A1-AR at more moderate levels (30-fold normal), which affords cardioprotection in the isolated heart while minimizing bradycardia and arrhythmia during ischemia in intact mice. Wild-type (WT; n ϭ 10) and moderate-level A1-AR TG (n ϭ 10) mice underwent 45 min of left anterior descending coronary artery occlusion, followed by 24-h reperfusion. Infarct size and region at risk were determined by triphenyltetrazolium chloride and phthalo blue staining, respectively. Infarct size (% region at risk) in WT mice was 52 Ϯ 3%, whereas overexpression of A1-ARs in the TG mice markedly reduced infarct size to 31 Ϯ 3% (P Ͻ 0.05). Furthermore, contractile function (left ventricular ejection fraction) as determined by cardiac magnetic resonance imaging 24 h after MI was better preserved in TG vs. WT mice. Cardiac overexpression of A1-ARs reduces infarct size by 40% and preserves cardiac function in intact mice after MI.ischemia-reperfusion; cardioprotection; transgenic mice; magnetic resonance imaging REPETITIVE A 1 -adenosine receptor (A 1 -AR) activation can maintain the heart in a protected state against myocardial ischemia-reperfusion injury (6). However, prolonged activation of A 1 -ARs with pharmacological agents induces a state of tolerance that blunts this cardioprotective effect (25). One possible explanation for this tolerance is the desensitization of A 1 -ARs resulting from continuous stimulation by A 1 -AR agonists. Using transgenic techniques, we showed (11-15, 19, 21) that cardiac A 1 -AR overexpression activates endogenous protective mechanisms that provide the heart with increased resistance to ischemia-reperfusion injury. Unlike transient ischemic preconditioning that occurs in wild-type (WT) animals, the constitutive preconditioning secondary to transgenic overexpression of the A 1 -AR has the potential to provide continuous cardioprotection (21). Although our past work was critical in establishing the long-term cardioprotective potential of A 1 -AR overexpression, these studies were conducted in isolated hearts from a line of mice with a 300-fold level of overexpression. In this study, we sought to determine whether A 1 -AR overexpression could also protect intact mice against ischemiareperfusion injury.Although cardiac-specific 300-fold A 1 -AR overexpression provided cardioprotection in globally ischemic, isolated heart models of ischemia-reperfusion injury, this was associated with adverse side effects such as significant resting bradycardia [h...

We tested the hypothesis that myocardial ischemia-reperfusion (I/R)-induced apoptosis is attenuated in transgenic mice overexpressing cardiac A(1) adenosine receptors. Isolated hearts from transgenic (TG, n = 19) and wild-type (WT, n = 22) mice underwent 30 min of ischemia and 2 h of reperfusion, with evaluation of apoptosis, caspase 3 activity, function, and necrosis. I/R-induced apoptosis was attenuated in TG hearts. TG hearts had less I/R-induced apoptotic nuclei (0.88 +/- 0.10% vs. 4.22 +/- 0.24% terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells in WT, P< 0.05), less DNA fragmentation (3.30 +/- 0.38-fold vs. 4.90 +/- 0.39-fold over control in WT, P < 0.05), and less I/R-induced caspase 3 activity (145 +/- 25% over nonischemic control vs. 234 +/- 31% in WT, P < 0.05). TG hearts also had improved recovery of function and less necrosis than WT hearts. In TG hearts pretreated with LY-294002 (3 microM) to evaluate the role of phosphosinositol-3-kinase in acute signaling, there was no change in the functional protection or apoptotic response to I/R. These data suggest that cardioprotection with transgenic overexpression of A(1) adenosine receptors involves attenuation of I/R-induced apoptosis that does not involve acute signaling through phosphoinositol-3-kinase.

A(3) adenosine receptors (A(3)ARs) have been implicated in regulating mast cell function and in cardioprotection during ischemia-reperfusion injury. The physiological role of A(3)ARs is unclear due to the lack of widely available selective antagonists. Therefore, we examined mice with targeted gene deletion of the A(3)AR together with pharmacological studies to determine the role of A(3)ARs in myocardial ischemia-reperfusion injury. We evaluated the functional response to 15-min global ischemia and 30-min reperfusion in isovolumic Langendorff hearts from A(3)AR(-/-) and wild-type (A(3)AR(+/+)) mice. Loss of contractile function during ischemia was unchanged, but recovery of developed pressure in hearts after reperfusion was improved in A(3)AR(-/-) compared with wild-type hearts (80 +/- 3 vs. 51 +/- 3% at 30 min). Tissue viability assessed by efflux of lactate dehydrogenase was also improved in A(3)AR(-/-) hearts (4.5 +/- 1 vs. 7.5 +/- 1 U/g). The adenosine receptor antagonist BW-A1433 (50 microM) decreased functional recovery following ischemia in A(3)AR(-/-) but not in wild-type hearts. We also examined myocardial infarct size using an intact model with 30-min left anterior descending coronary artery occlusion and 24-h reperfusion. Infarct size was reduced by over 60% in A(3)AR(-/-) hearts. In summary, targeted deletion of the A(3)AR improved functional recovery and tissue viability during reperfusion following ischemia. These data suggest that activation of A(3)ARs contributes to myocardial injury in this setting in the rodent. Since A(3)ARs are thought to be present on resident mast cells in the rodent myocardium, we speculate that A(3)ARs may have proinflammatory actions that mediate the deleterious effects of A(3)AR activation during ischemia-reperfusion injury.

Selective A(3)AR activation is cardioprotective in wild-type hearts and hearts overexpressing A(1)ARs, yet A(3)AR gene deletion generates an ischemia-tolerant phenotype without altering energy metabolism or pH. This may be due to compensatory changes or undefined genotypic differences in A(3)AR KO vs. wild-type hearts.

A(1) adenosine receptor overexpression is associated with a decreased rate of active Ca(2+) transport into the SR. We hypothesize that changes in SR function may cause a depletion of the SR Ca(2+) pool, which might protect from ischemic injury by delaying the development of cytosolic Ca(2+) overload during ischemia.

Myocardial damage from ischemia/reperfusion injury can be minimized through endogenous protective mechanisms, including activation of A 1 adenosine receptors. Transgenic mice with cardiac-specific A 1 adenosine receptor overexpression (A 1 AR Trans) have demonstrated functional and metabolic tolerance to in vitro and in vivo myocardial ischemia/reperfusion injury. The purpose of this investigation is to examine the role of p38 MAP kinase in A 1 receptor-mediated cardioprotection. Activation of p38 MAPK by A 1 adenosine receptor stimulation was evaluated in neonatal rat cardiomyocytes subjected to simulated ischemia. A 1 activation by cyclopentyladenosine decreased cell death and simultaneously enhanced p38 phosphorylation by simulated ischemia. The role of p38 MAP kinase activation in cardioprotection with A 1 adenosine receptor overexpression was evaluated in isolated perfused hearts from A 1 AR Trans mice subjected to 20 min ischemia and 30 min reperfusion. Inhibition of p38 MAPK activity with 10 mM SB-203580 prior to ischemia and during reperfusion decreased postischemic total functional recovery in A 1 AR Trans hearts from 54 7 1 to 2777% of maximal and increased final EDP from 1073 mmHg to 3478 mmHg. A 1 adenosine receptor-mediated activation of p38 MAP kinase in cardiomyocytes is involved in the protection of cells from ischemic death. Tolerance to ischemic injury by A 1 receptor overexpression is dependent upon activation of p38 MAPK. These findings suggest that adenosine A 1 mediated cardioprotection in transgenic mice involves activation of the p38 MAP kinase cascade. Drug Dev.