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Cardiovascular Research 2002 53(1):147-155; doi:10.1016/S0008-6363(01)00424-2
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Copyright © 2001, European Society of Cardiology

Effects of A3 adenosine receptor activation and gene knock-out in ischemic-reperfused mouse heart

Glenn J Harrisona, Rachael J Cerniwayb, Jason Pearta, Stuart S Berrc, Kevin Ashtona, Sara Reganb, G Paul Matherneb and John P Headricka,*

aHeart Foundation Research Centre, Griffith University Gold Coast Campus, Southport, QLD Australia
bDepartment of Pediatrics and the Cardiovascular Research Center, University of Virginia Health Sciences Center, Charlottesville, VA, USA
cDepartment of Radiology, University of Virginia Health Sciences Center, Charlottesville, VA, USA

* Corresponding author. Tel.: +61-7-5552-8292; fax: +61-7-5552-8802 j.headrick{at}mailbox.gu.edu.au

Received 21 March 2001; accepted 30 July 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Perfused heart preparation
 4. Experimental protocol
 5. 31P-NMR spectroscopic...
 6. Analysis of ATP...
 7. Chemicals
 8. Statistical analysis
 9. Results
 10. Discussion
 11. Conclusions
 References
 
Objectives: To characterize effects of A3 adenosine receptor (A3AR) activation and gene knock-out on responses to ischemia-reperfusion in mouse heart. Methods: Perfused hearts from wild-type and A3AR gene knock-out (A3AR KO) mice were subjected to 20 min ischemia and 30 min reperfusion. Functional responses were assessed and changes in energy metabolism and cytosolic pH monitored via 31P-NMR spectroscopy. Results: Selective A3AR agonism with 100 nM 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (chloro-IB-MECA) enhanced post-ischemic contractile recovery without altering contracture development in wild-type hearts, an effect unrelated to non-selective activation of A1 or A2 adenosine receptors. Chloro-IB-MECA also improved recovery in hearts overexpressing A1ARs. Paradoxically, post-ischemic recovery was enhanced by A3AR KO. Developed pressure, +dP/dt, and –dP/dt all recovered to higher levels in A3AR KO (70–80% of pre-ischemia) vs. wild-type hearts (45–50% of pre-ischemia) (P<0.05). Enhanced recovery was unrelated to recoveries of ATP, phosphocreatine (PCr), inorganic phosphate (Pi), energy state ([ATP]/[ADP]. [Pi], {Delta}GATP) or cytosolic pH. Conclusions: Selective A3AR activation is cardioprotective in wild-type hearts and hearts overexpressing A1ARs, yet A3AR 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 A3AR KO vs. wild-type hearts.

KEYWORDS Adenosine; Gene expression; Ischemia; NMR; Receptors; Reperfusion; Stunning


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Perfused heart preparation
 4. Experimental protocol
 5. 31P-NMR spectroscopic...
 6. Analysis of ATP...
 7. Chemicals
 8. Statistical analysis
 9. Results
 10. Discussion
 11. Conclusions
 References
 
Adenosine receptor activation reduces reversible and irreversible myocardial injury following ischemia-reperfusion. Mechanisms by which adenosine receptors protect are incompletely defined, and the precise roles of different receptor sub-types (A1, A2A, A2B, and A3 adenosine receptors) remain unclear. Most research regarding cardioprotection has focused on A1 adenosine receptor (A1AR) mediated protection, since this was thought to be the only receptor sub-type expressed in myocytes. However, there is now evidence that A2A adenosine receptors (A2AARs) are expressed in ventricular myocytes where they may reduce stunning and irreversible injury in post-ischemic myocardium [1,2]. Furthermore, A3ARs are also expressed in cardiomyocytes, and activation of these receptors ameliorates ischemic or hypoxic injury in isolated myocytes [3,4] and intact hearts [5–8].

Curiously, although a number of investigators document cardioprotective responses to A3AR agonism in different models [3–8], preliminary reports indicate that targeted deletion of A3ARs enhances ischemic tolerance and that A3ARs are not necessary in preconditioning [9–11]. Moreover, there is evidence that A3ARs may facilitate myocardial apoptosis [12,13] and worsen ischemic injury in other tissues [14]. To further define the role of A3ARs in cardiac ischemia-reperfusion we assessed effects of A3AR KO on: (i) functional tolerance, (ii) cellular energy state, and (iii) cytosolic pH, during global normothermic ischemia-reperfusion. Proposed mechanisms by which adenosine receptors may modify ischemic tolerance include modulation of substrate and energy metabolism and H+ handling [15–18].

We also studied the functional effects of selective A3AR agonism in wild-type mice to contrast effects of gene deletion with those of exogenous A3AR activation. No prior studies have examined the effects of A3AR activation during ischemia-reperfusion in the increasingly studied mouse heart.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Perfused heart preparation
 4. Experimental protocol
 5. 31P-NMR spectroscopic...
 6. Analysis of ATP...
 7. Chemicals
 8. Statistical analysis
 9. Results
 10. Discussion
 11. Conclusions
 References
 
The following investigations conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Homozygous A3AR–/– mice and wild-type A3AR+/+ litter-mate controls were provided by Merck Research Laboratories. Details of the generation and characterization of the A3AR–/– mice have been published previously [19]. For the various studies, hearts were obtained from mature 22–30 week old male and female (A3AR+/+) mice (n=9, 31.4±1.8 g body weight, 130±14 mg wet heart weight), homozygous A3AR null-mutant (A3AR–/–) mice (n=8, 30.5±1.6 g body weight, 128±6 mg wet heart weight), wild-type C57/Bl6 mice (n=58, 31.0±2.4 g body weight, 124±10 mg wet heart weight), and C57/Bl6 mice overexpressing cardiac A1ARs (n=16, 27.7±2.8 g body weight, 116±12 mg wet heart weight). Generation and properties of transgenic hearts overexpressing cardiac A1ARs have been described by us previously [18,20,21].


   3. Perfused heart preparation
Top
 Abstract
 1. Introduction
 2. Methods
 3. Perfused heart preparation
4. Experimental protocol
 5. 31P-NMR spectroscopic...
 6. Analysis of ATP...
 7. Chemicals
 8. Statistical analysis
 9. Results
 10. Discussion
 11. Conclusions
 References
 
Mice were anesthetized with 50 mg/kg sodium pentobarbital administered intraperitoneally, and hearts perfused as described previously [18,20,21]. After anesthesia a thoracotomy was performed and hearts rapidly excised into ice-cold perfusion fluid. The aorta was cannulated and the coronary circulation of all hearts perfused in a non-recirculating mode at a pressure of 80 mmHg with modified Krebs–Henseleit perfusion fluid containing (in mM): NaCl, 118; NaHCO3, 25; KCl, 4.7; KH2PO4, 1.2; CaCl2, 2.5; MgSO4, 1.2; glucose, 11; and EDTA, 0.6. Perfusion fluid was equilibrated with 95% O2, 5% CO2 at 37°C to give a pH of 7.4 and PO2 of ~550 mmHg at the tip of the aortic cannula. Perfusion fluid delivered to the heart was passed through a 0.45 µ Sterivex-HV filter cartridge (Millipore, Bedford, MA, USA). The left ventricle was vented with an apical drain and hearts instrumented for functional measurements (see below). They were then placed in a plastic chamber which was continuously superfused with warmed buffer, bathing the heart and maintaining temperature at 37°C. The perfusion chamber was surrounded by a dual-tuned 3-turn solenoidal 1H/31P NMR coil for NMR spectroscopy.

Contractile function was assessed via intra-ventricular isovolumic balloon, as described by us previously [20,21]. A fluid-filled balloon, constructed of pliable polyvinyl chloride plastic film (Saran WrapTM) tied with a 4-0 suture onto a short length of polyethylene tubing, was inserted into the left ventricle via the mitral. The film was initially stretched over a syringe tip to give a spherical form to the balloon. Balloons were 7–9 mm in deflated length and yielded pressures of less than 2 mmHg at an inflated volume of 60 µl. The fluid-filled balloons were connected to a P23 XL pressure transducer (Viggo-Spectramed, Oxnard, CA, USA) permitting continuous measurement of left ventricular pressure. Volume was increased by no more than 40 µl using a calibrated Hamilton 500 µl threaded plunger syringe (Hamilton, Reno, NV, USA) to give an end-diastolic pressure of 5 mmHg prior to ischemia. The balloon remained inflated throughout all experiments. Coronary flow was monitored via a cannulating Doppler flow-probe in the aortic perfusion line and connected to a T106 flowmeter (Transonic Systems, Ithaca, NY, USA). Functional data were recorded at 1 KHz on a 4-channel MacLab (AD Instruments, Castle Hill, Australia). The ventricular pressure signal was digitally processed to yield systolic pressure, diastolic pressure, +dP/dt, –dP/dt, and heart rate. The rate–pressure product was calculated as left ventricular developed pressurexheart rate. Hearts were excluded after 30 min stabilization if they met one of the following criteria: (i) coronary flow≥5 ml.min–1 (maximal dilation, or due to an aortic tear), (ii) unstable (fluctuating) contractile function, (iii) left ventricular systolic pressure below 100 mmHg, or (iv) significant arrhythmias. This amounted to less than 4% of all hearts prepared.


   4. Experimental protocol
Top
 Abstract
 1. Introduction
 2. Methods
 3. Perfused heart preparation
 4. Experimental protocol
5. 31P-NMR spectroscopic...
 6. Analysis of ATP...
 7. Chemicals
 8. Statistical analysis
 9. Results
 10. Discussion
 11. Conclusions
 References
 
For NMR studies the chamber housing hearts was introduced into an NMR magnet and after 30 min stabilization baseline measurements were made. Hearts were then subjected to 20 min of global normothermic ischemia followed by 30 min reperfusion. This protocol was performed in wild-type (n=9) and A3AR KO hearts (n=8).

To identify effects of selective A3AR activation in wild-type C57/Bl6 hearts we assessed effects of 100 nM chloro-IB-MECA (n=8), a selective A3AR agonist with a Ki of ~1 nM at the A3AR and 500–1000 nM at A2A and A1 receptors [12]. Chloro-IB-MECA at 100 nM produced no detectable changes in heart rate, contractile function or flow in pre-ischemic hearts. We repeated these experiments in the presence of 100 nM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an A1AR selective antagonist (n=7), or 200 nM 9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)(1,2,4)-triazolo[1,5-c]quinazoline (MRS-1220), an A3AR selective antagonist (n=7). We also examined the ability of A3AR agonism to modify contractile recovery in hearts overexpressing A1ARs. We have shown these hearts to be protected from ischemic-reperfusion [18,20,21], though it is unclear whether the level of tolerance is maximal in this model [20]. Transgenic hearts were subjected to 20 min ischemia and 30 min reperfusion in the absence (n=8) and presence of 100 nM chloro-IB-MECA (n=8). Finally, we compared effects of chloro-IB-MECA to those for 10 µM adenosine (n=9), 20 nM of A1AR selective N6-cyclopentyladenosine (CPA, n=8), and 1 nM of A2AAR selective 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680, n=9). CPA and CGS21680 concentrations were determined from concentration–response curves in preliminary studies. CPA at 20 nM produced 80% maximal bradycardia with no coronary dilation, and 1 nM CGS21680 induced 80% maximal dilation with no other functional changes (data not shown). Adenosine at 10 µM induced similar changes in heart rate and flow. Vehicle (control, n=10) and agonist treated hearts were perfused as described and were switched to ventricular pacing at 400 beats.min–1 10 min prior to induction of ischemia [18,20,21]. Pacing eliminated bradycardia with CPA and adenosine, normalizing heart rate between groups. After 5 min of pacing agonists or vehicle (0.005% DMSO) were infused to achieve the concentrations indicated. After a further 5 min global ischemia was initiated for 20 min followed by 30 min reperfusion. Pacing was terminated during ischemia and re-instated after 2 min reperfusion [20,21] as preliminary studies revealed that electrical pacing throughout ischemia exaggerates ischemic injury and enhanced post-ischemic arrhythmogenesis (data not shown).


   5. 31P-NMR spectroscopic analysis
Top
 Abstract
 1. Introduction
 2. Methods
 3. Perfused heart preparation
 4. Experimental protocol
 5. 31P-NMR spectroscopic...
6. Analysis of ATP...
 7. Chemicals
 8. Statistical analysis
 9. Results
 10. Discussion
 11. Conclusions
 References
 
31P-NMR data were acquired as described in detail by us for mouse hearts [18] and other species [15,22]. Hearts were introduced into the 40-cm bore of a Varian SIS 200/400 4.7 Tesla magnet (Varian Associates, Palo Alto, CA, USA) and the magnet shimmed on the heart and perfusion fluid 1H signal. 31P-spectra were acquired at 80.984 Mhz with spectra consisting of 120 signal averaged free induction decays (FIDs) acquired over 5-min periods (90° pulse, 2.0 s interpulse delay). FIDs were multiplied by a 25 Hz line broadening factor and spectral width was 4 KHz, consisting of 4096 data points. Intensities for β-ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) were determined by integration using resident Varian VNMR software, and were corrected for partial saturation using T1 values of 0.9±0.1, 2.2±0.3, and 1.9±0.2 s, respectively, acquired in wild-type mice via the inversion-recovery technique (n=5). Corrected intensities were normalized to chemically determined total tissue ATP content measured in a sub-set of wild-type mice (see below) [18].

Cytosolic pH and intracellular free [Mg2+] were calculated from chemical shifts of Pi relative to PCr, and the relative {alpha}-P and β-P shifts of ATP, as described in detail recently [18]. The phosphorylation ratio was calculated as [ATP]/[ADP].[Pi], with free cytosolic [ADP] estimated from the creatine kinase equilibrium:

Formula
where creatine (Cr) was determined by subtraction of PCr from total creatine content, and K'ck is the observed creatine kinase equilibrium constant corrected for pH and Mg2+ [22]. Free cytosolic [5'-AMP] was determined from the adenylate kinase equilibrium:

Formula
where K'ak is the observed adenylate kinase equilibrium constant corrected for intracellular pH and Mg2+ [18,22]. The Gibbs free energy of ATP hydrolysis ({Delta}GATP) was calculated as:

Formula
where R is the ideal gas constant (8.314 J.K–1.mole–1), T is temperature in Kelvin (310 K), and the standard free energy of ATP hydrolysis ({Delta}G°ATP) is calculated as –RTln K'ATP, and K'ATP is the observed equilibrium constant for ATPase [18,22].


   6. Analysis of ATP and total creatine content
Top
 Abstract
 1. Introduction
 2. Methods
 3. Perfused heart preparation
 4. Experimental protocol
 5. 31P-NMR spectroscopic...
 6. Analysis of ATP...
7. Chemicals
 8. Statistical analysis
 9. Results
 10. Discussion
 11. Conclusions
 References
 
Myocardial ATP and total creatine pool (PCr+creatine) concentrations were determined in perchloric acid extracts from control hearts freeze-clamped at liquid N2 temperature (n=5). Samples were analyzed for ATP, PCr and Cr by HPLC as described previously [18,22]. Tissue content in mmol.g–1 wet weight was converted to mM intracellular water based on an intracellular volume of 0.5 ml.g–1 wet weight. ATP and PCr+creatine levels determined in this way were 8.0±0.6 and 25.6±3.1 mM, respectively.


   7. Chemicals
Top
 Abstract
 1. Introduction
 2. Methods
 3. Perfused heart preparation
 4. Experimental protocol
 5. 31P-NMR spectroscopic...
 6. Analysis of ATP...
 7. Chemicals
8. Statistical analysis
 9. Results
 10. Discussion
 11. Conclusions
 References
 
All chemicals were of analytical grade or better. Chloro-IB-MECA, CPA, CGS21680, DPCPX, and MRS-1220 were all purchased from Sigma/RBI (St Louis, MO, USA).


   8. Statistical analysis
Top
 Abstract
 1. Introduction
 2. Methods
 3. Perfused heart preparation
 4. Experimental protocol
 5. 31P-NMR spectroscopic...
 6. Analysis of ATP...
 7. Chemicals
 8. Statistical analysis
9. Results
 10. Discussion
 11. Conclusions
 References
 
Data are expressed as mean±S.E.M. Functional variables were compared by one-way analysis of variance. Metabolic profiles were assessed via two-way analysis of variance for repeated measures. When significance was detected a Newman–Keuls post-hoc analysis was employed for individual comparisons. In all tests significance was accepted for P<0.05.


   9. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Perfused heart preparation
 4. Experimental protocol
 5. 31P-NMR spectroscopic...
 6. Analysis of ATP...
 7. Chemicals
 8. Statistical analysis
 9. Results
10. Discussion
 11. Conclusions
 References
 
9.1 Normoxic function and responses to ischemia in wild-type and A3AR KO hearts
There were no differences in normoxic contractile function between wild-type, A3AR KO, and transgenic hearts overexpressing A1ARs, although heart rate prior to pacing was reduced in the latter group (Table 1). As documented previously [18,20,21], global normothermic ischemia rapidly inhibited contractile function in all hearts (within 2–4 min).


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  		Table 1 Baseline functional parameters in perfused mouse hearts from wild-type mice (n=9), A3AR KO mice (n=8), and transgenic mice overexpressing cardiac A1ARs (n=8)

 
Ischemic contracture was rapid and pronounced, and there were no differences in contracture development between wild-type and A3AR KO hearts (Fig. 1A). Diastolic and developed pressures failed to recover fully during reperfusion in both groups (Fig. 1). A3AR KO did not alter initial recovery of contractile function or coronary flow (Fig. 1B). However, final recoveries for left ventricular developed pressure, +dP/dt, –dP/dt, and the rate–pressure product were all significantly improved in A3AR KO hearts (Fig. 1C). Coronary flow ultimately recovered to pre-ischemic levels in both groups (Fig. 1C).


Figure 1
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  		Fig. 1 Responses to ischemia and reperfusion in wild-type hearts (n=9) and A3AR KO hearts (n=8). Data is shown for (A) time to ischemic contracture (TIC) and peak ischemic contracture during 20 min global ischemia, (B) immediate post-ischemic functional recoveries for left ventricular end-diastolic pressure (EDP), left ventricular diastolic pressure (LVDP) +dP/dt, –dP/dt, the rate–pressure product (RPP), and coronary flow, measured at 2 min of reperfusion, and (C) final post-ischemic recoveries for EDP, LVDP, +dP/dt, –dP/dt, RPP, and coronary flow measured at 30 min of reperfusion. Recovery of EDP is shown in absolute units while all other recoveries are expressed as % of pre-ischemia. Values shown are means±S.E.M. *P<0.05 vs. wild-type hearts.

 
9.2 Metabolic response to ischemia-reperfusion in wild-type and A3AR KO hearts
31P-NMR spectroscopy revealed no differences in metabolic responses between wild-type and A3AR KO hearts (Fig. 2). Ischemia reduced ATP, PCr, pH and energy state, followed by significant recovery during reperfusion (Figs. 2 and 3Go). There was a sustained post-ischemic elevation in Pi levels, reduction in ATP, and depression of the phosphorylation ratio in both groups (Figs. 2 and 3Go). Though not shown, free cytosolic ADP and AMP levels increased dramatically with ischemia and fell to levels approaching pre-ischemia on reperfusion. There were no differences in cytosolic levels of these metabolites between A3AR KO and wild-type hearts at any time (data not shown). The phosphorylation ratio and {Delta}GATP responded similarly in wild-type A3AR KO hearts (Fig. 3).


Figure 2
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  		Fig. 2 Changes in myocardial ATP, PCr, Pi, and cytosolic pH during 20 min global normothermic ischemia and 30 min reperfusion in wild-type (n=9) and A3AR KO hearts (n=8). Values shown are means±S.E.M. *P<0.05 vs. pre-ischemia. Note that all metabolite values measured during the 20 min ischemic period differed significantly from pre-ischemia (P<0.05). There were no differences in metabolites between wild-type and A3AR KO hearts at any time point.

 

Figure 3
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  		Fig. 3 Cellular energy state indicated by the cytosolic phosphorylation ratio ([ATP]/[ADP].[Pi]) and Gibbs free energy of ATP hydrolysis ({Delta}GATP) during 20 min global normothermic ischemia and 30 min reperfusion in wild-type (n=9) and A3AR KO hearts (n=8). Values shown are means±S.E.M. *P<0.05 vs. pre-ischemia. Note that all values measured during the 20-min ischemic period differed significantly from pre-ischemic values (P<0.05). There were no differences between wild-type and A3AR KO hearts at any time.

 
9.3 Functional effects of selective adenosine receptor agonism in wild-type hearts and in hearts overexpressing A1ARs
To identify effects of A3AR activation in wild-type hearts we assessed effects of 100 nM chloro-IB-MECA, together with the effects of A1AR and A2AAR activation with CPA and CGS21680, respectively, and non-selective receptor activation with adenosine itself. None of the receptor agonists altered pre-ischemic contractile function, with all functional variables remaining at 98–102% of pre-infusion levels (data not shown). Neither chloro-IB-MECA nor CPA altered coronary flow (95–98% of pre-infusion) whereas CGS21680 significantly increased flow to 34.2±1.9 ml.min–1.g–1 (P<0.05), and adenosine increased pre-ischemic flow to 39.8±2.0 ml.min–1.g–1 (P<0.05). Post-ischemic recovery was not altered by CPA or CGS21680. In contrast, adenosine and chloro-IB-MECA both significantly enhanced post-ischemic contractile recovery, as indicated by left ventricular developed pressure, +dP/dt, and –dP/dt (Fig. 4A). Coronary re-flow was not altered by CPA or chloro-IB-MECA (Fig. 4A).


Figure 4
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  		Fig. 4 (A) Effects of A1AR, A2AAR or A3AR agonism on post-ischemic functional recovery after 20 min ischemia and 30 min reperfusion (expressed as % of pre-ischemic function in control or treated hearts). (B) Effects of A3AR agonism on post-ischemic functional recovery after 20 min ischemia and 30 min reperfusion in transgenic hearts overexpressing A1ARs. Recoveries for left ventricular diastolic pressure (LVDP) (sqdiagf), +dP/dt ({square}), –dP/dt ({blacksquare}), and coronary flow ({image}) were acquired in vehicle treated wild-type C57/Bl/6 hearts (n=10), in wild-type hearts treated with either 10 µM adenosine (n=9), 20 nM CPA (n=8), 1 nM CGS21680 (n=9), 100 nM chloro-IB-MECA (n=8), 100 nM chloro-IB-MECA+100 nM DPCPX (n=7), and 100 nM chloro-IB-MECA+200 nM MRS-1220 (n=7), and in hearts overexpressing A1ARs which were either untreated (n=8) or treated with 100 nM chloro-IB-MECA (n=8). Values shown are means±S.E.M. * P<0.05 vs. control or wild-type hearts; {dagger} P<0.05 MRS1220 or DPCPX treated vs. untreated hearts; {ddagger} P<0.05 chloro-IB-MECA treated vs. untreated transgenic hearts.

 
While re-flow, expressed as a percentage of pre-ischemic flow, was unaltered by CGS21680 and adenosine (Fig. 4A), coronary flow remained significantly higher during reperfusion in CGS21680 (27.8±1.8 ml.min–1.g–1 after 30 min) and adenosine-treated (31.9±2.9 ml.min–1.g–1) vs. untreated hearts (20.7±3.3 ml.min–1.g–1) (P<0.05). Infusion of 100 nM DPCPX did not modify protective effects of chloro-IB-MECA in wild-type hearts (Fig. 4A). In contrast, 200 nM MRS-1220 reversed protection due to chloro-IB-MECA. Rate and extent of ischemic contracture were not altered by any of the agonists studied (data not shown).

As documented previously [18,20,21], transgenic overexpression of A1ARs significantly enhanced post-ischemic contractile recovery during reperfusion (Fig. 4B).

Recovery in transgenic hearts was equal to or superior to that in chloro-IB-MECA treated wild-type hearts (Fig. 4). Treatment of transgenic hearts with 100 nM chloro-IB-MECA further enhanced post-ischemic contractile recovery. Ischemic contracture development was significantly reduced by A1AR overexpression, and unaltered by A3AR agonism in these hearts (data not shown).


   10. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Perfused heart preparation
 4. Experimental protocol
 5. 31P-NMR spectroscopic...
 6. Analysis of ATP...
 7. Chemicals
 8. Statistical analysis
 9. Results
 10. Discussion
11. Conclusions
 References
 
The goals of this study were to characterize functional and metabolic effects of A3AR knock-out in ischemic-reperfused murine myocardium, and to compare these effects with those for A3AR agonism in wild-type hearts. Our data indicate that A3AR agonism enhances ischemic tolerance in wild-type hearts and hearts overexpressing A1ARs. Paradoxically, A3AR KO also apparently enhances functional tolerance without altering energy metabolism or H+ handling. Since we do not directly address effects of A3ARs on necrosis or apoptosis we cannot determine how much of the improved functional recovery is due to reduced cell death. Nonetheless, our data demonstrate paradoxical improvement of ischemic tolerance with selective A3AR agonism and in a genetically modified A3AR KO line.

10.1 Functional and metabolic effects of A3AR KO
Hearts from A3AR KO mice exhibited enhanced tolerance to ischemia-reperfusion (Fig. 1), largely due to improved systolic rather than diastolic function. This supports the notion that A3AR activation can be injurious [12,13,23], and preliminary [9,10] and recent observations [11] regarding the A3AR KO phenotype. Since previous studies demonstrate that adenosine mediated cardioprotection is associated with improved energy metabolism and H+ handling [15–18], we assessed metabolic responses via 31P-NMR spectroscopy. 31P-NMR data reveal that A3AR KO does not improve functional tolerance via such metabolic actions (Figs. 2 and 3Go). However, post-ischemic recoveries of ATP, Pi and energy state were incomplete in wild-type and KO hearts (Figs. 2 and 3Go), potentially contributing to impaired post-ischemic function [18].

10.2 Functional protection via A3AR agonism
Since no data exist regarding effects of A3AR agonists in mouse heart, and since gene deletion data are not directly applicable to wild-type tissues, we examined effects of A3AR agonism in wild-type hearts and in hearts overexpressing A1ARs (Fig. 4). A3AR activation was beneficial in both groups, consistent with prior studies in rat, rabbit and porcine myocardium [3–8,12,24]. Protection was not associated with improved re-flow. A3AR activation also failed to modify contracture development, in contrast with the protective effects of A1AR activation by endogenous adenosine [15–17] and of A1AR overexpression [18,20,21].

While chloro-IB-MECA is highly selective for A3AR vs. A1 or A2AARs in other species [12], there are no data regarding its Ki at murine receptors. Though the agonist failed to alter heart rate (sensitive to A1ARs) and coronary flow (sensitive to A2AARs), it is feasible that protection occurs via A1 or A2AAR activation. However, CPA and CGS21680, both >100-fold selective for A1ARs and A2AARs, respectively, failed to modify post-ischemic recovery at 80% maximally effective concentrations (Fig. 4). This contrasts with protection from chloro-IB-MECA, which did not exert detectable A1AR or A2AR mediated responses.

Furthermore, effects of chloro-IB-MECA were comparable to those for adenosine, were unaltered by A1AR antagonism, and were reversed by A3AR antagonism with MRS-1220 (Fig. 4). These data, and >1000-fold A3AR selectivity of chloro-IB-MECA in other species [14], indicate that non-selective effects of this A3AR agonist cannot explain protection.

A potential explanation for similar effects of A3AR activation and A3AR knock-out stems from ‘paradoxical’ effects of A3AR activation [12]. Low levels of A3AR activation may reduce injury whereas high levels enhance apoptosis and mast cell degranulation [12,23]. However, this cannot explain our observations — if low or tonic A3AR activation reduced injury while high levels of activation enhance injury, A3AR agonists and A3AR deletion would both impair rather than improve ischemic tolerance.

10.3 Lack of protection with A1AR and A2AAR agonism
Lack of protection with A1AR agonism is consistent with some studies [21,25,26], but contrasts with others [27–32]. Interestingly, many of the studies demonstrating protection employed A1AR agonist concentrations which will activate A2ARs and A3ARs. N6-cyclohexyladenosine attenuates ischemic injury at concentrations 250- to 500-fold higher than its functional EC50 at A1ARs [28–30]. While 0.1–1.0 µM R-phenylisopropyl-adenosine [30,33,36] and 0.2 µM 2-chloro-N6-cyclopentyladenosine are cardioprotective [31], these levels are up to 30-fold higher than Kis at A3ARs [19,33]. CPA, used in the present study, is 100-fold selective for A1ARs (Ki 0.3–1.0 nM) vs. A3ARs (Ki 30–200 nM) [6,33]. We employed a concentration producing 80% maximal A1AR-mediated bradycardia and below its Ki at A3ARs. This selective concentration fails to modify ischemic tolerance. In contrast an equipotent concentration of adenosine was protective, as discussed previously [20,21,25],

A1AR-mediated protection is normally near maximal during ischaemia, when A1ARs exert a major portion of their protective effects [21,27]. Selective A1AR agonism should therefore be ineffective (Fig. 4), whereas A1AR overexpression should provide protection [20,25], as shown in Fig. 4B. Interestingly, A3AR agonism further protects the latter hearts, demonstrating that the pronounced protection with A1AR overexpression is not maximal, as suggested previously [20]. Moreover, since effects of A3AR agonism are additive to those of A1AR overexpression, our data support differing pathways by which A1ARs and A3ARs can mediate cardioprotection.

Lack of A2AAR-mediated protection is consistent with studies in isolated hearts [28], and the notion that A2AARs protect via modulating inflammatory processes in blood [1,35]. Although there is some support for A2AAR-mediated protection in isolated hearts [36,37], these data were acquired in hearts from different species perfused at low aortic pressures and coronary flows. Consequently the hearts possessed poor contractile function prior to ischemia, rendering comparison with our findings and other studies [28,34] difficult.

10.4 Study limitations
Paradoxical protection in A3AR KO hearts despite A3AR-mediated protection in wild-type hearts may reflect limitations inherent in gene knock-out studies. Studies of gene ‘deletion’ are necessarily limited to assessing effects of a genes absence rather than its normal function. Caution must be used in extrapolation to wild-type tissue. A3ARs may be important in cardiovascular development and function, and gene inactivation may induce morphological or physiological abnormalities complicating interpretation. While A3AR KO mice are fertile and visually and histologically comparable to wild-type mice [19], unknown compensatory mechanisms may be activated in the absence of A3ARs, clouding interpretation. Indeed, the general paucity of lethal gene deletions shows considerable genetic redundancy (compensation) must exist. Both positive and negative effects of gene deletion can be attributed to the absence of the gene and/or to compensatory changes in one or more other paths. Investigations of compensation may actually prove to be a more valuable use of knock-out models [38].

With respect to the A3AR KO phenotype, Zhao et al. [39] recently showed that cardiovascular cAMP is elevated. As discussed by Lasley et al. [2], cAMP is depressed in stunned myocardium, agents enhancing cAMP improve post-ischemic function, and elevated cAMP may play a role in preconditioning. Thus, the ischemia-tolerant phenotype may result from elevated cAMP. Since Zhao et al. found that adenosine elevated cAMP in hearts and vessels of A3AR KO mice, it is also possible that protection could arise from exaggerated cAMP-mediated responses at A1ARs or A2ARs. However, A1ARs reduce rather than elevate cAMP, and neither A1 nor A2AAR activation is protective in this model (Fig. 4). Furthermore, while increased vascular cAMP [39] is predicted to enhance coronary hyperemia, Fig. 1 shows initial hyperemia and final recovery of coronary flow are unaltered by A3AR KO.

A key limitation is that phenotypic changes in knock-outs may stem from linked or ‘hitch-hiking’ genes [40]. A3AR KO mice were obtained by introduction of embryonic stem cells from 129 strain mice into blastocysts, with chimeric embryos raised and mated to wild-type C57BL/6 and B6D2 mice [19]. Subsequent matings generate wild-types, heterozygous null-mutant, and homozygous null-mutant mice with recombinant genotypes from different parental strains. Recombination patterns may differ between litter-mates and the chromosome with the targeted locus will carry 129-type alleles which tend to remain with the mutated A3AR allele [40]. In contrast, control mice are less likely to possess 129 alleles. The phenotype resulting from gene mutation may therefore arise from background 129 or ‘hitch-hiker’ genes rather than the targeted mutation itself. In support of this possibility, pronounced strain-related differences in myocardial ischemic tolerance have been documented in the rat [41], and SV129 strain mice have been shown to be more tolerant of cerebral ischemia than C57/Bl6 mice [42]. Thus, genotypic determinants of ischemic tolerance may differ between wild-types and A3AR KO mice containing different 129 genetic material.


   11. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Perfused heart preparation
 4. Experimental protocol
 5. 31P-NMR spectroscopic...
 6. Analysis of ATP...
 7. Chemicals
 8. Statistical analysis
 9. Results
 10. Discussion
 11. Conclusions
References
 
In summary, this study reveals that A3AR gene knock-out improves functional tolerance to ischemia without altering energy metabolism or acidosis. This contrasts with cardioprotection via selective A3AR agonism in wild-type hearts, a selective effect unrelated to A1 or A2AAR activation. We conclude that A3AR activation is protective in ischemic-reperfused mouse heart, yet A3AR knock-out yields a paradoxically ischemia-tolerant phenotype. The latter findings in A3AR KO hearts may reflect limitations in extrapolating gene knock-out data to wild-type tissues. A3AR knock-out may yield an ischemia-tolerant phenotype as a result of obscure compensatory changes, altered cAMP levels, and/or expression of linked genes.

Time for primary review 23 days.


   Acknowledgements
 
This work was supported by grants from the National Institutes of Health RO1 grant (HL 59419), the National Health and Medical Research Council of Australia, and the National Heart Foundation of Australia (G 98B 0080 and G 99B 0246).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Perfused heart preparation
 4. Experimental protocol
 5. 31P-NMR spectroscopic...
 6. Analysis of ATP...
 7. Chemicals
 8. Statistical analysis
 9. Results
 10. Discussion
 11. Conclusions
 References
 

  1. Jordan J.E., Zhao Z.Q., 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 (1997) 280:301–309.[Abstract/Free Full Text]
  2. Lasley R.D., Jahania M.S., Mentzer R.M. Jr. Beneficial effects of adenosine A2a agonist CGS-21680 in infarcted and stunned porcine myocardium. Am J Physiol Heart Circ Physiol (2001) 280:H1660–H1668.[Abstract/Free Full Text]
  3. Strickler J., Jacobson K.A., Liang B.T. Direct preconditioning of cultured chick ventricular myocytes. Novel functions of cardiac adenosine A2a and A3 receptors. J Clin Invest (1996) 98:1773–1779.[ISI][Medline]
  4. Parsons M., Young L., Lee J.E., Jacobson K.A., Liang B.T. Distinct cardioprotective effects of adenosine mediated by differential coupling of receptor subtypes to phospholipases C and D. FASEB J (2000) 14:1423–1431.[Abstract/Free Full Text]
  5. Auchampach J.A., Rizvi A., Qiu Y., Tang X.-L., Maldonado C., Teschner S., Bolli R. Selective activation of A3 adenosine receptors with N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide protects against myocardial stunning and infarction without hemodynamic changes in conscious rabbits. Circ Res (1997) 80:800–809.[Abstract/Free Full Text]
  6. Carr C.S., Hill R.J., Masamune H., Kennedy S.P., Knight D.R., Tracey W.R., Yellon D.M. Evidence for a role for both the adenosine A1 and A3 receptors in protection of isolated human atrial muscle against simulated ischaemia. Cardiovasc Res (1997) 36:52–59.[Abstract/Free Full Text]
  7. Hill R.J., Oleynek J.J., Magee W., Knight D.R., Tracey W.R. Relative importance of adenosine A1 and A3 receptors in mediating physiological or pharmacological protection from ischemic myocardial injury in the rabbit heart. J Mol Cell Cardiol (1998) 30:579–585.[CrossRef][ISI][Medline]
  8. Thourani V.H., Nakamura M., Ronson R.S., Jordan J.E., Zhao Z.Q., Levy J.H., Szlam F., Guyton R.A., Vinten-Johansen J. Adenosine A3-receptor stimulation attenuates postischemic dysfunction through KATP channels. Am J Physiol Heart Circ Physiol (1999) 277:H228–H235.[Abstract/Free Full Text]
  9. Jones R., Morrison R.R., Jacobson M.A., Matherne G.P. Targeted deletion of adenosine A3 receptors increases myocardial resistance to ischemia-reperfusion injury. Circulation (1999) 100:3775. Abstract.
  10. Auchampach J.A., Bolli R., Bao W., Wu W.J., Black R.G., Hobson L., Salvatore C.A., Jacobson M.A., Guo Y. A3 receptor knock-out mice are resistant to ischemic injury and exhibit early ischemic preconditioning. Drug Dev Res (2000) 50:26. Abstract.
  11. Guo Y., Bolli R., Bao W., Wu W.-J., Black R.G. Jr., Murphree S.S., Salvatore C.A., Jacobson M.A., Auchampach J.A. Targeted deletion of the A3 adenosine receptor confers resistance to myocardial ischemic injury and does not prevent early preconditioning. J Mol Cell Cardiol (2001) 33:825–830.[CrossRef][ISI][Medline]
  12. Jacobson K.A. Adenosine A3 receptors: novel ligands and paradoxical effects. Trends Pharmacol Sci (1998) 19:184–191.[CrossRef][Medline]
  13. Shneyvays V., Jacobson K.A., Li A.H., Nawrath H., Zinman T., Isaac A., Shainberg A. Induction of apoptosis in rat cardiocytes by A3 adenosine receptor activation and its suppression by isoproterenol. Exp Cell Res (2000) 257:111–126.[CrossRef][ISI][Medline]
  14. Lee H.T., Emala C.W. Protective effects of renal ischemic preconditioning and adenosine pretreatment: role of A1 and A3 receptors. Am J Physiol Renal Physiol (2000) 278:F380–F387.[Abstract/Free Full Text]
  15. Angello D.A., Headrick J.P., Coddington N.M., Berne R.M. Adenosine antagonism attenuates metabolic but not functional recovery from ischemia. Am J Physiol Heart Circ Physiol (1991) 260:H193–H200.[Abstract/Free Full Text]
  16. Lasley R.D., Mentzer R.M. Jr. Protective effects of adenosine in the reversibly injured heart. Ann Thorac Surg (1995) 60:843–846.[Abstract/Free Full Text]
  17. Fralix T.A., Murphy E., London R.E., Steenbergen C. Protective effects of adenosine in the perfused rat heart: changes in metabolism and intracellular ion homeostasis. Am J Physiol Cell Physiol (1993) 264:C986–C994.[Abstract/Free Full Text]
  18. Headrick J.P., Gauthier N.S., Berr S.S., Morrison R.R., Matherne G.P. Transgenic A1 adenosine receptor overexpression improves myocardial energy state during ischemia-reperfusion. J Mol Cell Cardiol (1998) 30:1059–1064.[CrossRef][ISI][Medline]
  19. Salvatore C.A., Tilley S.L., Latour A.M., Fletcher D.S., Koller B.H., Jacobson M.A. Disruption of the A3 adenosine receptor gene in mice and its effects on stimulated inflammatory cells. J Biol Chem (2000) 275:4429–4434.[Abstract/Free Full Text]
  20. Morrison R.R., Jones R., Byford A.M., Stell A.R., Peart J., Headrick J.P., Matherne G.P. Transgenic overexpression of cardiac A1 adenosine receptors mimics ischemic preconditioning. Am J Physiol Heart Circ Physiol (2000) 279:H1071–H1078.[Abstract/Free Full Text]
  21. Peart J., Headrick J.P. Intrinsic activation of A1 adenosine receptors during ischemia and reperfusion improves ischemic tolerance. Am J Physiol Heart Circ Physiol (2000) 279:H2166–H2175.[Abstract/Free Full Text]
  22. Headrick J.P., McKirdy J.C., Willis R.J. Functional and metabolic effects of extracellular magnesium in normoxic and ischemic myocardium. Am J Physiol Heart Circ Physiol (1998) 275:H917–H929.[Abstract/Free Full Text]
  23. Linden J. Cloned adenosine A3 receptors: pharmacological properties, species differences and receptor functions. Trends Pharmacol Sci (1994) 15:298–306.[CrossRef][Medline]
  24. Thourani V.H., Ronson R.S., Jordan J.E., Guyton R.A., Vinten-Johansen J. Adenosine A3 pretreatment before cardioplegic arrest attenuates postischemic cardiac dysfunction. Ann Thorac Surg (1999) 67:1732–1737.[Abstract/Free Full Text]
  25. Matherne G.P., Linden J., Byford A.M., Gauthier N.S., Headrick J.P. Transgenic A1 adenosine receptor over-expression increases myocardial resistance to ischemia. Proc Natl Acad Sci USA (1997) 94:6541–6546.[Abstract/Free Full Text]
  26. Grover G.J., Baird A.J., Sleph P.G. Lack of pharmacological interaction between ATP-sensitive potassium channels and adenosine A1 receptors in ischemic rat hearts. Cardiovasc Res (1996) 31:511–517.[Abstract/Free Full Text]
  27. Lasley R.D., Rhee J.W., Van Wylen D.G.L., Mentzer R.M. Jr. Adenosine A1 receptor mediated protection of the globally ischemic isolated rat heart. J Mol Cell Cardiol (1990) 22:29–47.
  28. Lasley R.D., Mentzer R.M. Jr. Adenosine improves recovery of postischemic myocardial function via an adenosine A1 receptor mechanism. Am J Physiol Heart Circ Physiol (1992) 23:H1460–H1465.
  29. Lasley R.D., Mentzer R.M. Jr. Pertussis toxin blocks adenosine A1 receptor mediated protection of the ischemic rat heart. J Mol Cell Cardiol (1993) 25:815–821.[CrossRef][ISI][Medline]
  30. Fraser H., Lopaschuk G.D., Clanachan A.S. Alteration of glycogen and glucose metabolism in ischaemic and post-ischaemic working rat hearts by adenosine A1 receptor stimulation. Br J Pharmacol (1999) 128:197–205.[CrossRef][ISI][Medline]
  31. Narayan P., Mentzer R.M. Jr., Lasley R.D. Adenosine A1 receptor activation reduces reactive oxygen species and attenuates stunning in ventricular myocytes. J Mol Cell Cardiol (2001) 33:121–129.[CrossRef][ISI][Medline]
  32. Miura T., Liu Y., Kita H., Ogawa T., Shimamoto K. Roles of mitochondrial ATP-sensitive K+ channels and PKC in anti-infarct tolerance afforded by adenosine A1 receptor activation. J Am Coll Cardiol (2000) 35:238–245.[Abstract/Free Full Text]
  33. Franchetti P., Cappellacci L., Marchetti S., Trincavelli L., Martini C., Mazzoni M.R., Lucacchini A., Grifantini M. 2'-C-Methyl analogues of selective adenosine receptor agonists: synthesis and binding studies. J Med Chem (1998) 41:1708–1715.[CrossRef][ISI][Medline]
  34. Finegan B.A., Lopaschuk G.D., Gandhi M., Clanachan A.S. Inhibition of glycolysis and enhanced mechanical function of working rat hearts as a result of adenosine A1 receptor stimulation during reperfusion following ischaemia. Br J Pharmacol (1996) 118:355–363.[ISI][Medline]
  35. Zhao Z.Q., Todd J.C., Sato H., Ma X.L., Vinten-Johansen J. Adenosine inhibition of neutrophil damage during reperfusion does not involve KATP-channel activation. Am J Physiol Heart Circ Physiol (1997) 273:H1677–H1687.[Abstract/Free Full Text]
  36. Cargnoni A., Ceconi C., Boraso A., Bernocchi P., Monopoli A., Curello S., Ferrari R. Role of A2A receptor in the modulation of myocardial reperfusion damage. J Cardiovasc Pharmacol (1999) 33:883–893.[CrossRef][ISI][Medline]
  37. Lozza G., Conti A., Ongini E., Monopoli A. Cardioprotective effects of adenosine A1 and A2A receptor agonists in the isolated rat heart. Pharmacol Res (1997) 35:57–64.[CrossRef][ISI][Medline]
  38. Crawley J.N. Unusual behavioral phenotypes of inbred mouse strains. Trends Neurosci (1996) 19:181–182.[CrossRef][ISI][Medline]
  39. Zhao Z., Makaritsis K., Francis C.E., Gavras H., Ravid K. A role for the A3 adenosine receptor in determining tissue levels of cAMP and blood pressure: studies in knock-out mice. Biochim Biophys Acta (2000) 1500:280–290.[Medline]
  40. Gerlai R. Gene-targeting studies of mammalian behavior: is it the mutation or the background genotype? Trends Neurosci (1996) 19:177–181.[CrossRef][ISI][Medline]
  41. Baker J.E., Konorev E.A., Gross G.J., Chilian W.M., Jacob H.J. Resistance to myocardial ischemia in five rat strains: is there a genetic component of cardioprotection? Am J Physiol Heart Circ Physiol (2000) 278:H1395–H1400.[Abstract/Free Full Text]
  42. Wellons J.C., Sheng H.X., Laskowitz D.T., Mackensen G.B., Pearlstein R.D., Warner D.S. A comparison of strain-related susceptibility in two murine recovery models of global cerebral ischemia. Brain Res (2000) 868:14–21.[CrossRef][ISI][Medline]

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