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(Circulation Research. 2005;96:363.)
© 2005 American Heart Association, Inc.

Integrative Physiology

Genetic Deletion of the A₁ Adenosine Receptor Limits Myocardial Ischemic Tolerance

Melissa E. Reichelt^*, Laura Willems^*, Jose G. Molina, Chun-Xiao Sun, Janci C. Noble, Kevin J. Ashton, Jurgen Schnermann, Michael R. Blackburn, John P. Headrick

From the Heart Foundation Research Center (M.E.R., L.W., K.J.A., J.P.H.), Griffith University, Southport, Australia; the Department of Biochemistry and Molecular Biology (J.G.M., C.-X.S., J.C.N., M.R.B.), University of Texas Health Science Center at Houston, Medical School, Houston; and the National Institute of Diabetes and Digestive and Kidney Diseases (J.S.), National Institutes of Health, Bethesda, Md.

Correspondence to John Headrick, Heart Foundation Research Centre, Griffith University, Southport, QLD 4217, Australia. E-mail J.Headrick{at}griffith.edu.au

	Abstract

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Adenosine receptors may be important determinants of intrinsic ischemic tolerance. Genetically modified mice were used to examine effects of global A₁ adenosine receptor (A₁AR) knockout (KO) on function and ischemic tolerance in perfused mouse hearts. Baseline contractile function and heart rate were unaltered by A₁AR KO, which was shown to abolish the negative chronotropic effects of 2-chloroadenosine (A₁AR-mediated) without altering A₂ adenosine receptor–mediated coronary dilation. Tolerance to 25 minutes global normothermic ischemia (followed by 45 minutes reperfusion) was significantly limited by A₁AR KO, with impaired contractile recovery (reduced by 25%) and enhanced lactate dehydrogenase (LDH) efflux (increased by 100%). Functional effects of A₁AR KO involved worsened systolic pressure development with little to no change in diastolic dysfunction. In contrast, cardiac specific A₁AR overexpression enhanced ischemic tolerance with a primary action on diastolic dysfunction. Nonselective receptor agonism (10 µmol/L 2-chloroadenosine) protected wild-type and also A₁AR KO hearts (albeit to a lesser extent), implicating protection via subtypes additional to A₁ARs. However, A₁AR KO abrogated effects of 2-chloroadenosine on ischemic contracture and diastolic dysfunction. These data are the first demonstrating global deletion of the A₁AR limits intrinsic myocardial resistance to ischemia. Data indicate the function of intrinsically activated A₁ARs appears primarily to be enhancement of postischemic contractility and limitation of cell death.

Key Words: adenosine • A₁ adenosine receptor • gene knockout • ischemia • reperfusion

	Introduction

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The heart possesses protective or retaliatory mechanisms providing tolerance to ischemia/reperfusion. These may represent targets for therapeutic manipulation of ischemic tolerance, and conversely, alterations in these mechanisms could underlie changes in outcome with aging and/or disease. We and others have been studying the role of the purine nucleoside adenosine and its receptors in modulating injury during ischemia/reperfusion.^1,2 There are currently four known and potentially protective adenosine receptor (AR) subtypes encoded by distinct genes: the A₁, A_2A, A_2B, and A₃ARs.^1,2 All are G-coupled, with A₁, A_2A, and A₃ARs possessing higher affinities for adenosine, whereas the A_2BAR has a relatively low affinity. Although contentious,² the AR system may be an integral component of the hearts intrinsic protective arsenal, limiting damage during^3–5 and after ischemic challenge.⁶ Although this is consistent with benefit via AR agonists,^1–3,7 effects of AR antagonism (to unmask responses to endogenous adenosine) are equivocal, with studies supporting^4–6,8–11 and refuting^12–15 a role for endogenous adenosine in dictating ischemic tolerance. Some of this controversy may stem from inherent limitations in pharmacological approaches to abrogating receptor-mediated responses: these can be hampered by potentially poor antagonist selectivity or potency, and/or potentiation of local agonist levels as a result of opening feedback loops linking "signal" (adenosine generation in this case) to tissue "response" (protection of cellular homeostasis).^16–18 An alternative approach involves selective gene deletion, which, coupled with complementary analysis of effects of transgenic overexpression, may facilitate assessment of the specific role of a protein in wild-type tissue.^19,20 In this study, we document for the first time the ability of genetic removal of A₁ARs to modify intrinsic tolerance to ischemia.

	Materials and Methods

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Investigations conformed to 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).

Experimental Protocol
A₁AR knockout (A₁AR KO) mice (Jackson Laboratories, Bar Harbor, Me) were generated and genotyped as described previously,²¹ with genotypes determined via PCR analysis of genomic DNA. All mice were on a mixed 129sv/C57BL/6J background and phenotypic comparisons were performed among littermates. Details of generation of C57/BL/6J mice selectively overexpressing cardiac A₁ARs have been reported previously.²² Hearts for this study were isolated from young (2 month) mice from the following groups: A₁AR KO (n=20); wild-type littermates (n=24); A₁AR overexpression (n=15); and wild-type C57/BL/6J (n=16).

All mice were anesthetized with 50 mg/kg sodium pentobarbitone administered intraperitoneally, a thoracotomy performed, and hearts excised into ice-cold perfusion fluid for cannulation and perfusion on a Langendorff perfusion system.^5,23 Hearts were stabilized at intrinsic rate a further 10 minutes before acquiring concentration-response curves for 2-chloroadenosine–mediated A₁AR and A_2AAR-dependent bradycardia/coronary dilation. Concentration-response curves were acquired in unpaced normoxic hearts from A₁AR KO (n=6), wild-type littermates (n=7), A₁AR overexpressing mice (n=7), and wild-type C57/BL/6J mice (n=7), as described previously.²⁴ Chronotropic and vasodilatory responses were scaled as percentage of baseline, and data were analyzed via nonlinear regression to acquire individual pEC₅₀ values, as outlined previously.^24,25

In ischemic studies, hearts were stabilized for 20 minutes at intrinsic heart rate before pacing at 420 bpm followed by a further 10 minutes stabilization period.^5,23 Baseline measurements were then made, and hearts were subjected to 25 minutes global normothermic ischemia followed by 45 minutes aerobic reperfusion. Coronary venous effluent was collected on ice for enzymatic analysis of lactate dehydrogenase (LDH) activity.²³ Total LDH efflux during reperfusion was expressed as international units (IU) per gm wet weight, and has been previously shown to correlate with measures of oncotic injury/infarction in this model.²⁶ Ischemic responses were assessed in A₁AR KO (n=7) and wild-type littermate (n=9) mice, and A₁AR overexpressing (n=8) and wild-type littermate (n=9) mice.

Effects of adenosinergic cardioprotection with 10 µmol/L of the nonselective agonist 2-chloroadenosine were also assessed in wild-type (n=8) and A₁AR KO hearts (n=7) subjected to 25 minutes ischemia and 45 minutes reperfusion. Based on concentration-response data in Figure 1, >3 µmol/L 2-chloroadenosine is required to maximally activate a functional A₁AR response (less for an A₂AR response). Thus, in an attempt to achieve near-maximal activation of all adenosine receptor subtypes in all murine lines studied, we used a 10 µmol/L agonist concentration. Although it is feasible prolonged treatment with high concentrations of 2-chloroadenosine might induce adenosine receptor-independent actions (because it is a substrate for nucleoside transporters), this is unlikely to be an issue in the current acute studies. The 10 µmol/L concentration is equivalent to or less than functional EC₅₀ values for 2-chloroadenosine activation of adenosine receptor responses in cardiovascular and other cell types, ^27–31 recent studies confirm this concentration induces receptor-mediated actions in cardiomyocytes and cardiac fibroblasts (mimicked by selective receptor agonists and/or blocked by adenosine receptor antagonists),^32–35 and our preliminary experiments (data not shown) confirmed acute chronotropic and vasodilatory responses to 10 µmol/L 2-chloroadenosine were sensitive to 100 µmol/L of the competitive receptor antagonist 8-sulfophenyltheophylline.

View larger version (26K): [in this window] [in a new window]   Figure 1. Effects of A1AR KO and transgenic A1AR overexpression on cardiac and vascular sensitivity to adenosine receptor agonism with 2-chloroadenosine. A, A1AR-mediated bradycardia. B, A2AAR-mediated coronary dilation. Responses are shown for hearts from A1AR KO (n=6) and wild-type littermate (n=7) mice, and for hearts from transgenic (TG) mice overexpressing cardiac A1ARs (n=7) and their wild-type littermates (n=7). Values are mean±SEM. *P<0.05 vs corresponding wild type.

Statistical Analyses
All data are presented as mean±SEM. Baseline data, pEC₅₀ values, final recoveries, and LDH efflux were analyzed via one-way ANOVA. Time course data were compared via two-way ANOVA for repeated measures. When significant differences were detected in ANOVA tests, a Newman-Keuls post hoc test was used for specific comparisons. A value of P<0.05 was considered significant in all tests.

	Results

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There were no differences in baseline contractile function or coronary flow between groups (Table). However, intrinsic heart rate was reduced in A₁AR-overexpressing hearts. Concentration-response analysis confirmed A₁AR KO abrogates A₁AR-mediated bradycardia without altering sensitivity of A_2AAR-mediated vasodilation (Figure 1). Conversely, A₁AR overexpression increased the sensitivity of A₁AR-mediated bradycardia without altering coronary responses. The pEC₅₀ values for the different responses are provided in Table.

View this table: [in this window] [in a new window]   Table 1. Preischemic Functional Parameters and Sensitivities (pEC50s) for 2-Chloroadenosine–Mediated Bradycardia (A1AR-dependent) and Coronary Vasodilation (A2AR-dependent) in Hearts From Each Experimental Group

Deletion of A₁ARs significantly reduced ischemic tolerance. Recovery profiles for hearts subjected to 25 minutes ischemia and 45 minutes reperfusion are depicted in Figure 2. Effects of A₁AR KO were evident in terms of reduced systolic pressure with little change in diastolic dysfunction (Figure 2). Thus, left ventricular pressure development was depressed (Figures 2 and 3). Deletion of the A₁AR did not modify the rate of contracture development during ischemia (time to reach 20 mm Hg diastolic pressure)²³ (Figure 3B), but significantly worsened cellular damage indicated by postischemic efflux of LDH (Figure 3C). Conversely, A₁AR overexpression enhanced ischemic tolerance (Figures 2 and 3), with the primary contractile effect being reduced diastolic dysfunction (Figures 2A and 3A). Overexpression of A₁ARs only improved systolic function during the initial minutes of reperfusion (Figure 2B). Ischemic contracture development was also reduced by A₁AR overexpression (Figure 3B), in contrast to lack of effect of A₁AR KO on this parameter.

View larger version (22K): [in this window] [in a new window]   Figure 2. Effects of manipulation of A1AR expression on postischemic recoveries for left ventricular diastolic pressure (A), systolic pressure (B), and developed pressure (C). Data are shown for recoveries in hearts from A1AR KO (n=7) and wild-type (WT) littermate (n=9) mice, and for hearts from transgenic (TG) mice overexpressing cardiac A1ARs (n=8) and their wild-type littermates (n=9). Values are mean±SEM. *P<0.05 vs corresponding wild type.

View larger version (26K): [in this window] [in a new window]   Figure 3. Effects of manipulation of A1AR expression and the nonselective adenosine receptor agonist 2-chloroadenosine, on ischemic and postischemic outcomes. A, Final recoveries for left ventricular (LV) diastolic and developed pressure. B, Rate of ischemic contracture (time for diastolic pressure to reach 20 mm Hg during ischemia). C, Postischemic LDH efflux during 45 minutes reperfusion. Outcomes are shown for hearts from A1AR KO (n=7) and wild-type (WT) littermate (n=9) mice, and for hearts from transgenic (TG) mice overexpressing cardiac A1ARs (n=8) and their wild-type littermates (n=9). Effects of 10 µmol/L 2-chloroadenosine were assessed in hearts from A1AR KO (n=7) and wild-type littermate (n=8) mice. Values are mean±SEM. *P<0.05 vs corresponding wild-type groups; P<0.05 vs untreated hearts.

Treatment of wild-type and A₁AR KO hearts with the nonselective agonist 2-chloroadenosine improved postischemic outcomes in both groups (Figure 3). However, the protective actions of the agonist were significantly reduced in A₁AR KO versus wild-type hearts (Figure 3). Furthermore, beneficial effects of 2-chloroadenosine on diastolic dysfunction and ischemic contracture observed in wild-type hearts were abrogated by A₁AR KO (Figure 3B).

	Discussion

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The role of endogenous adenosine and adenosine receptors in determining intrinsic tolerance to ischemic insult remains controversial. Many studies do not observe effects of adenosine receptor antagonists on ischemic outcome in various species.^12–15 Moreover, there is even evidence A₁AR blockade actually improves outcome from ischemia.^36,37 In contrast, there is some support for a role for endogenously generated adenosine in protection of ischemic myocardium^4–6,8–11 and modulation of processes impinging on recovery from ischemia.^38,39 Several explanations may account for varied findings with antagonists, the most likely involving mixed selectivity and potency of agents used and the fact that an antagonist applied to any system in which the signal is coupled to the response (as with adenosine) will likely generate an elevation in the signal (ie, opening the feedback loop). This has been verified in prior work.^16–18 On the other hand, it is also important to note that generally observed cardioprotection with adenosine agonists^{1–3,8,10,26,40} indicates the intrinsic adenosine response must normally be submaximally (if at all) engaged. In assessing potential roles of adenosine receptors, an alternate and relatively selective approach involves gene deletion of receptor protein. In this study, we provide the first evidence that genetic deletion of the A₁AR significantly limits the ability of mouse myocardium to withstand injury during ischemia/reperfusion (Figures 2 and 3). Conversely, cardiac A₁AR overexpression confers enhanced tolerance, as documented previously.²² These data collectively provide strong support for a role of A₁ARs in determining intrinsic tolerance to ischemia/reperfusion.

Effects of A₁AR KO are evident in terms of reduced systolic dysfunction and oncotic injury, with little effect on diastolic dysfunction (Figures 2 and 3). This contrasts effects of A₁AR overexpression, which are manifest as reduced diastolic contracture with little change in systolic pressure (except during initial reperfusion). Thus, effects of receptor deletion do not mirror effects of receptor overexpression. Rather, data suggest responses mediated by a highly overexpressed receptor may be abnormal or "supraphysiological" and/or that functional effects of A₁ARs vary with the level of activation during insult. This is consistent with effects of 2-chloroadenosine, which did reduce postischemic diastolic dysfunction, an action ablated by A₁AR KO (Figure 3). These data indicate the A₁AR is responsible for "adenosinergic" reductions in diastolic dysfunction, but that the response is evident only with enhanced levels of agonism. Selective actions of A₁ARs on systolic versus diastolic function are consistent with prior observations regarding A₁AR antagonism, revealing that postischemic A₁AR activation improves systolic force without altering diastolic dysfunction, whereas intraischemic A₁AR activation limits diastolic dysfunction in addition to improving systolic force.⁵ Extent (and timing) of A₁AR engagement likely dictates relative effects on diastolic versus systolic dysfunction.

Development of ischemic contracture was also unaffected by A₁AR KO, but was limited by A₁AR overexpression and 2-chloroadenosine. The latter response was again abrogated by A₁AR KO (Figure 3). Thus, adenosinergic limitation of ischemic contracture is A₁AR dependent but evident only with exaggerated receptor agonism or expression. This agrees with early work of Lasley et al³ demonstrating A₁AR agonist–mediated protection against ischemic contracture in rat, and prior data demonstrating negligible effects of A₁AR blockade on contracture development in mice.⁵

Although 2-chloroadenosine–mediated protection against contracture and diastolic dysfunction is abrogated by A₁AR KO (Figure 3), supporting A₁AR-dependent effects of the agonist on diastolic function, the analogue still exerted some beneficial actions in A₁AR KO hearts. This is reflected in improved systolic function together with reduced LDH efflux (Figure 3). These effects, refractory to A₁AR KO, implicate a protective function for receptor subtypes distinct from A₁ARs. Prior evidence that exogenous A₃AR but not A_2AAR agonism is cardioprotective in the model studied here,^26,40 and that A₃AR protection selectively enhances systolic function and reduces cell death, argues for a potential role for this subtype in the remaining protection with 2-chloroadenosine. However, this remains to be directly assessed, and we cannot exclude a potential role for the less well-studied A_2BAR.

Two study limitations bear mention before closing. As with all gene deletion studies, adaptations may occur to compensate for life-long absence of a targeted protein. Although it may be fruitful to focus on such adaptations (see for example, Godecke et al⁴¹ and Warth and Barhanin⁴²), they also complicate interpretation of phenotypic outcomes. We have assessed, in part, obvious changes in other adenosine receptors, verifying that A₁AR deletion selectively abrogates an A₁AR response (bradycardia) without modifying A_2AAR sensitivity. However, we recognize the possibility of undetected compensatory changes contributing to the A₁AR KO phenotype. The second limitation relates to the fact that we focus on responses in the isolated buffer-perfused heart. This was deliberate, to assess more directly the myocardial phenotype, because A₁AR protection is primarily "direct" and mediated via cardiomyocyte receptors.^2,7 However, adenosinergic protection in vivo additionally involves modulation of blood cells and related inflammatory responses.^1,2,7 We therefore cannot ascertain potential effects of A₁AR deletion on these extracardiac responses. However, these responses are predominantly A₂AR-dependent^2,7 and thus not predicted to be substantially modified by A₁AR KO. In addition, intrinsic A₁AR-dependent protection during ischemia/reperfusion in vivo may involve actions of adenosine generated within neutrophils, platelets, and other blood-borne cells. Thus, this extracardiac component will be absent in the present model, potentially leading to an underestimation of the normal extent of A₁AR activation by endogenously generated adenosine.

In summary, the current analysis of the effects of A₁AR KO supports an important function of A₁ARs in dictating intrinsic myocardial resistance to ischemia/reperfusion. Effects of A₁AR deletion suggest these receptors normally play a role in enhancing postischemic contractility and limiting cell death, with little effect on abnormalities in diastolic function. Finally, reduced yet significant protection via the nonselective agonist 2-chloroadenosine in A₁AR KO hearts implicates a significant cardioprotective response mediated via adenosine receptors additional to the A₁AR.

	Acknowledgments

 
This work was supported by NIH AI-43572 (to M.R.B.) and National Health and Medical Research Council of Australia grant 231416 (to J.P.H.). J.P.H. was also the recipient of a career development fellowship from the National Heart Foundation of Australia. We are extremely grateful for the provision of mice overexpressing A₁ARs by Prof Paul Matherne and for the excellent technical assistance of Kirsten Holmgren.

	Footnotes

 
*Both authors contributed equally to this study.

Original received September 10, 2004; revision received January 4, 2005; accepted January 4, 2005.

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