Activation of myocardial A 1 adenosine receptors (A 1 AR) protects the heart from ischemic injury. In this study transgenic mice were created using the cardiac-specific ␣-myosin heavy chain promoter and rat A 1 AR cDNA. Heart membranes from two transgene positive lines displayed Ϸ1,000-fold overexpression of A 1 AR (6,574 ؎ 965 and 10,691 ؎ 1,002 fmol per mg of protein vs. 8 ؎ 5 fmol per mg of protein in control hearts). Compared with control hearts, transgenic Langendorff-perfused hearts had a significantly lower intrinsic heart rate (248 beats per min vs. 318 beats per min, P < 0.05), lower developed tension (1.2 g vs. 1.6 g, P < 0.05), and similar coronary resistance. The difference in developed tension was eliminated by pacing. Injury of control hearts during global ischemia, indexed by time-to-ischemic contracture, was accelerated by blocking adenosine receptors with 50 M 8-(p-sulfophenyl) theophylline but was unaffected by addition of 20 nM N 6 -cyclopentyladenosine, an A 1 AR agonist. Thus A 1 ARs in ischemic myocardium are presumably saturated by endogenous adenosine. Overexpressing myocardial A 1 ARs increased time-to-ischemic contracture and improved functional recovery during reperfusion. The data indicate that A 1 AR activation by endogenous adenosine affords protection during ischemia, but that the response is limited by A 1 AR number in murine myocardium. Overexpression of A 1 AR affords additional protection. These data support the concept that genetic manipulation of A 1 AR expression may improve myocardial tolerance to ischemia.

The role of A(1) adenosine receptors (A(1)AR) in ischemic preconditioning was investigated in isolated crystalloid-perfused wild-type and transgenic mouse hearts with increased A(1)AR. The effect of preconditioning on postischemic myocardial function, lactate dehydrogenase (LDH) release, and infarct size was examined. Functional recovery was greater in transgenic versus wild-type hearts (44.8 +/- 3.4% baseline vs. 25.6 +/- 1.7%). Preconditioning improved functional recovery in wild-type hearts from 25.6 +/- 1.7% to 37.4 +/- 2.2% but did not change recovery in transgenic hearts (44.8 +/- 3.4% vs. 44.5 +/- 3.9%). In isovolumically contracting hearts, pretreatment with selective A(1) receptor antagonist 1, 3-dipropyl-8-cyclopentylxanthine attenuated the improved functional recovery in both wild-type preconditioned (74.2 +/- 7.3% baseline rate of pressure development over time untreated vs. 29.7 +/- 7.3% treated) and transgenic hearts (84.1 +/- 12.8% untreated vs. 42.1 +/- 6.8% treated). Preconditioning wild-type hearts reduced LDH release (from 7,012 +/- 1,451 to 1,691 +/- 1,256 U. l(-1). g(-1). min(-1)) and infarct size (from 62.6 +/- 5.1% to 32.3 +/- 11.5%). Preconditioning did not affect LDH release or infarct size in hearts overexpressing A(1)AR. Compared with wild-type hearts, A(1)AR overexpression markedly reduced LDH release (from 7,012 +/- 1,451 to 917 +/- 1,123 U. l(-1). g(-1). min(-1)) and infarct size (from 62.6 +/- 5.1% to 6.5 +/- 2.1%). These data demonstrate that murine preconditioning involves endogenous activation of A(1)AR. The beneficial effects of preconditioning and A(1)AR overexpression are not additive. Taken with the observation that A(1)AR blockade equally eliminates the functional protection resulting from both preconditioning and transgenic A(1)AR overexpression, we conclude that the two interventions affect cardioprotection via common mechanisms or pathways.

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.

m1 Muscarinic signaling is inhibited by clinically relevant concentrations of lidocaine and by extracellularly administered QX314, suggesting that the major site of action is a extracellular domain of the muscarinic receptor. An additional less potent but superadditive inhibitory effect on the G-protein is suggested.

The advent of transgenic techniques provides a unique opportunity to study adenosine receptor‐mediated cardioprotection. Transgenic A1 receptor overexpression increases myocardial protection without impairing intrinsic function, but at the expense of resting bradycardia and a blunted response to catecholamine stimulation. Thus, we investigated whether an optimal degree of A1 receptor overexpression exists whereby hearts would be protected from ischemic injury with minimal sequelae. Hearts from two transgenic lines were evaluated (high‐level expressors, HL, and low‐level expressors, LL) and compared to controls. Both transgenic lines showed enhanced myocardial protection as measured by postischemic recovery of developed tension (HL 49 ± 3% of baseline, LL 45 ± 3%, and control 31 ± 3%; P < 0.05). Resting heart rates in conscious restrained mice were lowest in HL (620 ± 14 bpm), intermediate in LL (681 ± 11 bpm), and highest in control (713 ± 8 bpm), P < 0.05, while systolic blood pressure was similar in all groups. The inotropic response to isoproterenol was comparably blunted in HL and LL hearts (+dP/dt increased 33 ± 4% and 39 ± 3%, respectively) compared to control (58 ± 7%), P < 0.05. β‐adrenergic receptor density was upregulated only in the HL hearts (90 ± 12 fmol/mg) compared to LL and control (41 ± 9 and 32 ± 5, respectively, P < 0.05). In summary, both HL and LL hearts afforded cardioprotection from ischemia, with LL hearts exhibiting less bradycardia and less β‐adrenergic cross‐regulation. While the data suggest that genetic manipulation of receptor systems may provide a powerful tool to improve myocardial tolerance to injury, they also highlight the need for careful screening for effects on the integrated physiology of the system. Drug Dev. Res. 45:402–409, 1998. © 1998 Wiley‐Liss, Inc.

expression profile of mouse myocardium with transgenic overexpression of A1 adenosine receptors.

Myocardial A1 adenosine receptor (A1AR) overexpression protects hearts from ischemia-reperfusion injury; however, the effects during anoxia are unknown. We evaluated responses to anoxia-reoxygenation in wild-type (WT) and transgenic (Trans) hearts with approximately 200-fold overexpression of A1ARs. Langendorff perfused hearts underwent 20 min anoxia followed by 30 min reoxygenation. In WT hearts peak diastolic contracture during anoxia was 45+/-3 mmHg, diastolic pressure remained elevated at 18+/-3 mmHg after reoxygenation, and developed pressure recovered to 52+/-4% of pre-anoxia. A1AR overexpression reduced hypoxic contracture to 29+/-4 mmHg, and improved recovery of diastolic pressure to 8+/-1 mmHg and developed pressure to 76+/-3% of pre-anoxia. Mitochondrial K(ATP) blockade with 100 microM 5-hydroxydecanoate (5-HD) increased hypoxic contracture to 73+/-6 mmHg in WT hearts, reduced post-hypoxic recoveries of both diastolic (40+/-5 mmHg) and developed pressures (33+/-3 %). In contrast, 5-HD had no effect on hypoxic contracture (24+/-8 mmHg), or post-hypoxic diastolic (10+/-2 mmHg) and developed pressures (74+/-3%) in Trans hearts. In summary, (i) A1AR overexpression improves myocardial tolerance to anoxia-reoxygenation, (ii) intrinsic mitochondrial K(ATP) channel activation decreases hypoxic contracture and improves functional recovery in wild-type hearts, and (iii) mitochondrial K(ATP) channels do not appear to play a major role in the functional protection from anoxia afforded by A1AR overexpression.