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Cardiovascular Research 52 (2001) 120–129 www.elsevier.com / locate / cardiores Cardioprotection with adenosine metabolism inhibitors in ischemic–reperfused mouse heart a b b a, Jason Peart , G. Paul Matherne , Rachael J. Cerniway , John P. Headrick * a b Heart Foundation Research Centre, Griffith University Gold Coast Campus, Southport, QLD 4217 Australia Department of Pediatrics and the Cardiovascular Research Center, University of Virginia Health Sciences Center, Charlottesville, VA, USA Received 24 January 2001; accepted 28 May 2001 Abstract Objectives: To characterize the ‘anti-ischemic’ effects of adenosine metabolism inhibition in ischemic–reperfused myocardium. Methods: Perfused C57 / B16 mouse hearts were subjected to 20 min ischemia 40 min reperfusion in the absence or presence of adenosine deaminase inhibition (50 mM erythro-2-(2-hydroxy-3-nonyl)adenine; EHNA) adenosine kinase inhibition (10 mM iodotubercidin; IODO), or 10 mM adenosine. Hearts overexpressing A 1 adenosine receptors (A 1 ARs) were also studied. Results: EHNA treatment reduced ischemic contracture and post-ischemic diastolic pressure (1462 vs. 2061 mmHg), increased recovery of developed pressure (6663 vs. 5362%) and reduced LDH efflux (8.961.6 vs. 18.061.7 I.U. / g). IODO also improved functional recovery (to 6062%) and reduced LDH efflux (5.361.7 I.U. / g), as did treatment with 10 mM adenosine. Protection with EHNA was reversed by co-infusion of IODO or 50 mM 8-r-sulfophenyltheophylline (adenosine receptor antagonist), but unaltered by 20 mM inosine110 mm hypoxanthine. Similarly, effects of iodotubercidin were inhibited by EHNA and 8-r-sulfophenyltheophylline. A 1 AR overexpression exerted similar effects to EHNA and EHNA or IODO alone enhanced recovery while EHNA1IODO reduced recovery in transgenic hearts. Functional recoveries and xanthine oxidase reactant levels were unrelated in the groups studied. Conclusions: Adenosine deaminase or kinase inhibition protects from ischemia–reperfusion. Cardioprotection via these enzyme inhibitors requires a functioning purine salvage pathway and involves enhanced adenosine receptor activation. Reduced formation of inosine is unimportant in EHNA-mediated protection. 2001 Elsevier Science B.V. All rights reserved. Keywords: Adenosine; Contractile function; Ischemia; Reperfusion; Stunning 1. Introduction Extracellular adenosine may function as an endogenous cardioprotectant during ischemia–reperfusion [1,2]. Furthermore, myocardial adenosine formation, release and salvage are key determinants of ATP depletion and repletion following ischemic insult, and adenosine catabolism may be a source of damaging radicals via the xanthine oxidase reaction [2–4]. Due to adenosine’s central role, investigators have pursued adenosine catabolism modulation as a potential cardioprotective strategy. This research has focused on adenosine deaminase inhibitors [5–15], with no studies reporting effects of adenosine kinase *Corresponding author. Tel.: 161-7-5552-8292; fax: 161-7-55528908. E-mail address: [email protected] (J.P. Headrick). inhibition in ischemic heart. However, there is mixed support for the anti-ischemic effects of adenosine kinase inhibition in other tissues [16–18]. In relation to adenosine deaminase inhibition, the mechanisms underlying associated cardioprotection remain unclear. There is some support for reduced xanthine-oxidase-derived free radical formation and for improved maintenance of the adenine nucleotide pool [14,15]. While enhanced adenosine receptor activation must also play a role, the relative importance of these different mechanisms has not been determined. Interestingly, since xanthine oxidase activity is negligible in pig, rabbit and human hearts [3,4,19–21], reduced xanthine oxidase derived radicals should not contribute to protection with adenosine deaminase inhibition in these species [8,13,22–24]. Recent data also Time for primary review 34 days. 0008-6363 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0008-6363( 01 )00360-1 J. Peart et al. / Cardiovascular Research 52 (2001) 120 – 129 suggests that a significant proportion of reactants for xanthine oxidase are produced independently of adenosine deamination [25]. Given these various issues the present study was designed to define mechanisms underlying cardioprotection with adenosine metabolism inhibitors in ischemic–reperfused mouse myocardium. We reasoned that mechanisms of cardioprotection with adenosine metabolism inhibition could be unmasked by enhancing extracellular adenosine levels via: inhibition of adenosine deaminase (EHNA treatment); inhibition of adenosine phosphorylation (iodotubercidin treatment); inhibition of deamination and phosphorylation (EHNA1iodotubercidin treatment); inhibition of adenosine deaminase and adenosine receptor antagonism (EHNA18-r-sulfophenyltheophylline treatment); inhibition of adenosine phosphorylation and adenosine receptor antagonism (iodotubercidin18-r-sulfophenyltheophylline treatment); inhibition of adenosine deaminase in the presence of elevated inosine and hypoxanthine (EHNA1inosine / hypoxanthine); and infusion of exogenous adenosine itself. Cardioprotective effects of A 1 adenosine receptor overexpression were also compared to the effects of EHNA and iodotubercidin and the ability of adenosine metabolism inhibitors to protect transgenic hearts was tested. 2. Methods 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). Hearts were isolated from fed male and female wild-type C57 / BL6 mice (body weight5 27.560.6 g, heart weight513565 mg) and transgenic mice overexpressing A 1 ARs (body weight530.461.4 g, heart weight514969 mg). Transgenic mice overexpressing functionally coupled cardiac A 1 adenosine receptors by |100-fold have been extensively characterized and described by us in detail previously [26,27]. 121 range. Perfusate was filtered (5.0 mm) immediately after preparation and all perfusate delivered to the heart was passed through an in-line 0.45 mm Sterivex-HV filter cartridge (Millipore, Bedford, MA, USA) to continuously remove microparticulates. The left ventricle was vented with a polyethylene apical drain and hearts instrumented for functional measurements as described below. They were then immersed in warmed perfusate inside a water jacketed bath maintained at 378C. The temperature of perfusion fluid was monitored by a needle thermistor at the entry into the aortic cannula and the temperature of the water bath assessed using a second thermistor probe. Temperature was recorded using a three-channel Physitemp TH-8 digital thermometer (Physitemp Instruments, Clifton, NJ, USA). For assessment of isovolumic function fluid-filled balloons constructed of polyvinylchloride film were inserted into the left ventricle via the mitral valve. Balloons were connected to a P23 XL pressure transducer (Viggo-Spectramed, Oxnard, CA, USA) by fluid-filled polyethylene tube permitting continuous measurement of left ventricular pressure. Balloon volume was increased to give an enddiastolic pressure of |5 mmHg. Coronary flow was monitored via a cannulating Doppler flow-probe (Transonic Systems, Ithaca, NY, USA) in the aortic perfusion line connected to a T206 flowmeter (Transonic Systems). All functional data were recorded at 1 kHz on a 4 / s MacLab data acquisition system (ADInstruments, Castle Hill, Australia) connected to an Apple 7300 / 180 computer. The ventricular pressure signal was digitally processed to yield peak systolic pressure, diastolic pressure, 1dP/ dt, 2dP/ dt and heart rate. The rate–pressure product was calculated as the product of heart rate and left ventricular developed pressure. Hearts were excluded from the study after the 25 min stabilization period if they met one of the following functional criteria: (i) coronary flow $5 ml / min (near 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 2% of all hearts isolated. 2.1. Perfused heart preparation 2.2. Experimental protocol Mice were anesthetized with 60 mg / kg sodium pentobarbital administered intraperitoneally and perfused as described previously [26,27]. 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 at a pressure of 80 mmHg with modified Krebs–Henseleit buffer containing (in mM): NaCl, 120; NaHCO 3 , 25; KCl, 4.7; CaCl 2 , 2.5; MgCl 2 1.2; KH 2 PO 4 1.2, D-glucose, 15; and EDTA, 0.5. Perfusate was equilibrated with 95% O 2 –5% CO 2 at 378C to give a pH of 7.4 and pO 2 of |600 mmHg at the tip of the aortic cannula over a 1–5 ml / min flow After 25 min stabilization, hearts were switched to ventricular pacing at a rate of 7 Hz (Grass S9 stimulator, Quincy, MA, USA). Those hearts receiving drug treatment (EHNA, iodotubercidin, 8-r-sulfophenyltheophylline) were then treated for 10 min prior to induction of global normothermic ischemia. Baseline measurements were made and 20 min ischemia was initiated followed by 40 min reperfusion. Unless stated otherwise, drug infusions were reinstated on reperfusion. Pacing was terminated upon initiation of ischemia and resumed after 2 min of reperfusion in all groups [26,27]. In wild-type hearts there 122 J. Peart et al. / Cardiovascular Research 52 (2001) 120 – 129 were eight primary experimental groups: untreated (control, n514), or treated with 50 mM EHNA (n512), 10 mM iodotubercidin (n59), 50 mM EHNA110 mM iodotubercidin (n58), 50 mM 8-r-sulfophenyltheophylline (n58), 50 mM EHNA150 mM 8-r-sulfophenyltheophylline (n57), 10 mM iodotubercidin150 mM 8-r-sulfophenyltheophylline (n58), 10 mM adenosine (n58). Transgenic hearts overexpressing A 1 ARs were also studied. Transgenic hearts were subjected to 20 min ischemia and 40 min reperfusion. Responses were obtained for untreated transgenic hearts (n58), transgenic hearts treated with 50 mM EHNA (n57), transgenic hearts treated with 10 mM iodotubercidin (n58) and transgenic hearts treated with 50 mM EHNA110 mM iodotubercidin (n57). To examine the impact of adenosine catabolites on responses to EHNA, a subsequent series of experiments were performed in which effects of 20 mM inosine110 mM hypoxanthine (n58) were assessed in untreated (n5 8) and 50 mM EHNA treated hearts (n58). In these experiments inosine1hypoxanthine was infused for 10 min prior to global ischemia and during the initial 5 min of reperfusion. Maximally effective inhibitory levels of EHNA (50 mM) and iodotubercidin (10 mM) were determined based upon documented inhibitory properties of these compounds. EHNA has a Ki of |1 nM for adenosine deaminase and an IC 50 of 1–2 mM in intact cells [12] and 5 mM EHNA maximally inhibits myocardial adenosine deaminase in perfused hearts [28]. We therefore chose an EHNA concentration more than 10-fold higher than the reported IC 50 and |10-fold higher than levels shown to near maximally block the enzyme. With respect to iodotubercidin, Kroll et al. also showed that 50% maximal inhibition of the enzyme occurs at 0.05–0.10 mM iodotubercidin, with $1 mM iodotubercidin maximally inhibiting myocardial adenosine kinase activity [28]. We therefore chose a |10fold greater concentration to ensure near maximal inhibition. 2.3. Analysis of venous purine and enzyme efflux In some experimental groups coronary venous effluent was sampled and frozen at 2808C until analyzed by HPLC for adenosine, inosine, hypoxanthine, xanthine and uric acid, as outlined by us previously [29,30] but with the addition of a Waters photodiode array detector in place of a tunable UV detector. Normoxic efflux was calculated as the product of coronary flow (ml / min / g wet weight)3 effluent concentrations (nmol / ml). Early (0–5 min) and total post-ischemic purine efflux values were calculated as the product of concentration reached in the reperfusion effluent (nmol / ml)3reperfusion volume (ml / g wet weight). Similarly, lactate dehydrogenase (LDH) levels were also measured in the post-ischemic effluent of key experimental groups using a commercially available en- zymatic assay (Sigma, St. Louis, MO, USA) optimized for sensitivity. Total efflux during 40 min reperfusion is reported (I.U. / min / g). 2.4. Chemicals EHNA, iodotubercidin, adenosine and 8-r-sulfophenyltheophylline (RBI, Natick, MA, USA) were all dissolved directly in perfusion fluid and infused through a 0.22-mm filter at not more than 1% of coronary flow to achieve the final concentrations indicated. 2.5. Statistical analysis Data are expressed as mean6S.E.M. Functional responses to ischemia–reperfusion, time to contracture, peak ischemic contracture, purine efflux values and post-ischemic LDH efflux values were compared by a one-way analysis of variance. When significance was detected a Tukey’s post-hoc test was employed for individual comparisons. In all tests significance was accepted for P,0.05. We estimated the power of the analysis of variance (f ), based on the data for percentage recovery of rate–pressure product and time to ischemic contracture ]]]]]]]]] f 5œ(k 2 1) ? (groups MS 2 s 2 ) /k ? s 2 where k is the number of experimental groups, MS the 2 mean square and s the variance. This revealed less than a 5% chance of committing a Type 2 error when comparing these variables between the treatment groups. 3. Results 3.1. Normoxic function and purine metabolism in mouse myocardium Data for normoxic function in treated and untreated hearts are given in Table 1. Preischemic function was similar in all groups except for elevations in coronary flow in those hearts treated with EHNA, iodotubercidin or adenosine. There was a trend towards increased inotropic and lusitropic state (1dP/ dt, 2dP/ dt) with simultaneous EHNA plus iodotubercidin treatment and towards reduced inotropic and lusitropic state in hearts treated with 8-rsulfophenyltheophylline. However, these effects did not achieve significance (Table 1). The normoxic (and post-ischemic) purine efflux is presented in Table 2. Use of 50 mM EHNA increased normoxic adenosine efflux 3-fold from |1 to over 4 nmol / min / g. This occurred without a significant change in total purine efflux, being balanced by a decline in formation of inosine and its catabolites. Iodotubercidin alone increased adenosine release 6-fold to more than 8 nmol / min / g and treatment with 10 mM iodotubercidin and 50 J. Peart et al. / Cardiovascular Research 52 (2001) 120 – 129 123 Table 1 Baseline functional parameters in perfused mouse hearts untreated or treated with 50 mM EHNA, 10 mM iodotubercidin, 50 mM EHNA110 mM iodotubercidin, 50 mm 8-r-sulfophenyltheophylline, 50 mM EHNA150 mM 8-r-sulfophenyltheophylline, or adenosine a Control (n514) Diastolic pressure (mmHg) Rate–pressure product (mmHg/min31000) 1dP/dt (mmHg/s) 2dP/dt (mmHg/s) Coronary flow (ml/min/g) EHNA (n512) IODO (n58) EHNA1IODO (n58) 8-SPT (n58) EHNA18-SPT (n57) 562 261 462 362 63.861.4 71.562.4 72.262.5 72.262.8 64.262.8 65.462.5 63.361.6 68.562.3 70646487 67616578 68136272 81076380 54206429* 73656347 68616213 66316296 249806445 244866310 239736117 255976415 236706181* 250126306 238966100 44786145 27.661.3* 39.961.8* 35.461.6* 20.161.6 35.662.3* 39.862.1* 18.561.4 563 Adenosine (n58) 662 21.361.1 563 IODO18-SPT (n58) 362 a All parameters were measured after 30 min of aerobic perfusion. Values are means6S.E.M. *, P,0.05, vs. control. Note that hearts were electrically paced at 420 beats / min in all groups. mM EHNA together further increased adenosine efflux to 12 nmol / min / g without significantly altering efflux of other metabolites (Table 2). Effects of EHNA and EHNA plus iodotubercidin on purine efflux indicate that 40–50% of inosine and its catabolites are produced independently of adenosine deamination (i.e. derive from IMP dephosphorylation). adenosine efflux. Adenosine efflux was greater than that with either EHNA or iodotubercidin alone. Effects of EHNA and EHNA plus iodotubercidin on purine efflux demonstrate that 30–40% of inosine and its catabolites are produced independently of adenosine deamination (i.e. from IMP dephosphorylation) in post-ischemic myocardium. 3.2. Purine metabolism in post-ischemic mouse myocardium 3.3. Ischemic contracture development Purine efflux was significantly enhanced in post-ischemic hearts (Table 2). Though not shown, the majority of purine efflux was lost from hearts during the initial 5–10 min of reperfusion. EHNA treatment increased postischemic adenosine efflux |3-fold without substantially altering total purine efflux. Treatment with 10 mM iodotubercidin alone doubled adenosine efflux and increased total purine efflux during reperfusion (Table 2). Addition of EHNA and iodotubercidin together did not alter post-ischemic efflux of total purine beyond that observed with EHNA alone, but significantly increased Global normothermic ischemia rapidly abolished contractile function in all hearts within 150 s. Ischemic contracture in untreated hearts was rapid (|330 s) and pronounced (|100 mmHg peak pressure) (Fig. 1). Treatment with 50 mM EHNA significantly prolonged time to ischemic contracture and reduced peak contracture development. Adenosine alone reduced contracture development whereas iodotubercidin did not significantly modify contracture. Effects of EHNA on contracture were inhibited by adenosine receptor antagonism with 8-r-sulfophenyltheophylline and by adenosine kinase inhibition with iodotubercidin. Interestingly, although iodotubercidin Table 2 Purine efflux from normoxic and post-ischemic mouse hearts untreated or treated with 50 mM EHNA, 10 mM iodotubercidin, 50 mM EHNA110 mM iodotubercidin, sulfophenyltheophylline, or adenosine a Control (n514) Preischemia Adenosine (nmol / min / g) Inosine1hypoxanthine (nmol / min / g) o Purines (nmol / min / g) 0–40 min reperfusion Adenosine (nmol / min / g) Inosine1hypoxanthine (nmol / min / g) o Purines (nmol / min / g) 1.260.3 2.660.2 8.060.6 26.563.5 † 51.765.7 † 106.169.5 † EHNA (n512) IODO (n58) EHNA1IODO (n58) 4.260.6* 1.360.1* 9.161.7 8.460.7* 6.560.4* 17.861.0* 12.061.6* 1.460.3* 20.661.8* 76.6610.2* † 16.262.7* † 108.1611.1 † 33.365.6 † 80.766.4* † 135.0620.2 † 99.8611.8* † 17.762.2* † 146.4615.6* † a Purine efflux rates are shown under normoxic conditions and over the total 40 min reperfusion period following 20 min global normothermic ischemia. o Purines represents the sum of effluxes for adenosine1inosine1hypoxanthine1xanthine1uric acid. Hearts were untreated, or treated with 50 mM EHNA, 10 mM iodotubercidin, or 50 mM EHNA110 mM iodotubercidin. Values shown are means6S.E.M. *, P,0.05 vs. untreated hearts; †, P,0.05 vs. pre-ischemia. 124 J. Peart et al. / Cardiovascular Research 52 (2001) 120 – 129 Fig. 1. Effects of EHNA and iodotubercidin on rate (left axis) and extent of ischemic contracture development (right axis) during 20 min global ischemia. Hearts were untreated (Control, n514), or received 50 mM EHNA (n512), 10 mM iodotubercidin (Iodo, n58), 50 mM EHNA110 mM iodotubercidin (EHNA1Iodo, n58), 50 mM 8-r-sulfophenyltheophylline (8-SPT, n58), 50 mM EHNA150 mM 8-r-sulfophenyltheophylline (EHNA18-SPT, n57), 10 mM iodotubercidin150 mM 8-rsulfophenyltheophylline (Iodo18-SPT, n58), or 10 mM adenosine (Ado, n58). Values shown are means6S.E.M. *, P,0.05 vs. untreated hearts; †, P,0.05 cotreatment vs. individual metabolism inhibitor treatment. alone did not significantly alter contracture development, coinfusion of 8-r-sulfophenyltheophylline accelerated contracture in this treatment group. 3.4. Functional recovery from ischemia Upon reperfusion diastolic pressure fell to |30 mmHg during the first minute, rose to |45 mmHg after 5 min and then gradually declined to a value of 20 mmHg at the end of reperfusion (Fig. 2). The pattern of recovery for left ventricular developed pressure was similar, with an early peak to |50% of pre-ischemia at 1–2 min before a subsequent fall to a minimum of |10% at 5 min, followed by a gradual recovery throughout the remaining reperfusion period. Coronary flow initially recovered to a slightly higher value than in pre-ischemic hearts (data not shown) followed by a gradual decline to final values insignificantly lower than pre-ischemia. EHNA treatment substantially reduced post-ischemic diastolic dysfunction and increased recovery of pressure development (Fig. 2). Treatment with iodotubercidin also significantly improved functional recovery from ischemia, reducing diastolic dysfunction and improving pressure development. Post-ischemic coronary flow was not significantly altered by EHNA but was substantially elevated by iodotubercidin. Infusion of 10 mM adenosine exerted cardioprotection similar to that with EHNA or iodotubercidin alone. Inhibition of adenosine receptors with 50 mM 8-r-sulfophenyltheophylline partially reversed cardioprotective effects of both EHNA and iodotubercidin. Coinfusion of EHNA plus iodotubercidin reduced functional recovery to less than that observed with either EHNA or iodotubercidin alone. Infusion of elevated inosine (20 mM) plus hypoxanthine (10 mM) prior to ischemia and for the initial 5 min of Fig. 2. Effects of EHNA and iodotubercidin on functional recovery from 20 min global ischemia and 40 min reperfusion. Recoveries were determined for: (A) left ventricular diastolic pressure (mmHg); (B) rate–pressure product (mmHg / min and % pre-ischemia) and (C) coronary flow (ml / min / g and % pre-ischemia). Hearts were untreated (Control, n514), or received 50 mM EHNA (n512), 10 mM iodotubercidin (Iodo, n58), 50 mM EHNA110 mM iodotubercidin (EHNA1Iodo, n58), 50 mM 8-r-sulfophenyltheophylline (8-SPT, n58), 50 mM EHNA150 mM 8-r-sulfophenyltheophylline (EHNA18-SPT, n57), 10 mM iodotubercidin150 mM 8-r-sulfophenyltheophylline (Iodo18-SPT, n5 8), or 10 mM adenosine (Ado, n58). Values shown are means6S.E.M. *, P,0.05 vs. untreated hearts; †, P,0.05 cotreatment vs. individual metabolism inhibitor treatment. reperfusion had no effect on post-ischemic functional recovery or on the cardioprotective actions of EHNA. Specifically, infusion of inosine plus hypoxanthine failed to alter pre-ischemic contractile function (rate–pressure product573.863.0 and 72.362.8 in control or EHNAtreated hearts, respectively; P.0.05 vs. untreated hearts). Post-ischemic recovery of contractile function was unaltered by inosine plus hypoxanthine (5162% recovery of rate–pressure product) and this treatment failed to alter protection with EHNA (6361% recovery of rate–pressure J. Peart et al. / Cardiovascular Research 52 (2001) 120 – 129 product). Treatment with inosine plus hypoxanthine also failed to alter recoveries for coronary flow (data not shown). 3.5. Responses to ischemia in transgenic hearts overexpressing A1 adenosine receptors Left ventricular function and coronary flow was similar in transgenic hearts compared with wild-type hearts. Preischemic rate–pressure product was 60.462.4 mmHg / 125 min31000 and coronary flow was 24.562.6 ml / min / g. Normoxic adenosine efflux in transgenic hearts was 1.460.3 nmol / min / g and total normoxic purine efflux was 7.061.1 nmol / min / g. Use of 50 mM EHNA increased adenosine efflux to 3.860.6 nmol / min / g (P,0.05). None of these efflux values differed from those for untreated or EHNA-treated wild-type hearts shown in Table 2. Average post-ischemic adenosine and total purine effluxes over 40 min reperfusion were 762 and 3667 nmol / min / g, respectively. EHNA significantly increased postischemic adenosine efflux to 3466 nmol / min / g (P,0.05) without substantially altering total purine efflux (4365 nmol / min / g). These values were significantly lower than corresponding values in wild-type hearts (Table 2). A 1 AR overexpression exerted cardioprotection which was similar to that observed with EHNA or iodotubercidin infusion (Fig. 3). All strategies significantly reduced postischemic diastolic dysfunction and increased relative recoveries for ventricular developed pressure (Figs. 2 and 3). EHNA significantly enhanced functional recovery from ischemia in transgenic hearts, as did treatment with iodotubercidin. Contractile function in these hearts almost recovered to pre-ischemic levels. Coinfusion of EHNA plus iodotubercidin did not alter recovery of transgenic hearts from ischemia (Fig. 3). 3.6. Post-ischemic enzyme leakage Pre-ischemic LDH leakage from perfused mouse hearts was less than 0.05 I.U. / min / g. The rate of enzyme leakage was markedly enhanced by ischemia, with |18 I.U. / g lost from control hearts over 40 min reperfusion (Fig. 4A). Post-ischemic LDH efflux tended to mirror changes in functional recovery with the various treatments. LDH leakage was significantly reduced by 60–70% by EHNA, iodotubercidin and adenosine (Fig. 4A). Beneficial effects of EHNA on LDH efflux were abolished by cotreatment with either iodotubercidin or 8-r-sulfophenyltheophylline. Transgenic A 1 AR overexpression also reduced post-ischemic LDH leakage (Fig. 4B). EHNA further reduced LDH leakage in the transgenic hearts while iodotubercidin produced an insignificant decline in enzyme loss. Fig. 3. Effects of transgenic overexpression of A 1 ARs on functional recovery from 20 min global normothermic ischemia and 40 min reperfusion. Recoveries were determined for (A) left ventricular diastolic pressure (mmHg); (B) rate–pressure product (mmHg / min and % preischemia) and (C) coronary flow (ml / min / g and % pre-ischemia). Transgenic hearts were untreated (Transgenic, n58), treated with 50 mM EHNA (Trans1EHNA, n57), treated with 10 mM iodotubercidin (Trans1Iodo, n58), or treated with 50 mM EHNA110 mM iodotubercidin (Trans1EHNA / Iodo, n57). Data for untreated wild-type hearts subjected to 20 min ischemia are also shown for comparison (n514). Values shown are means6S.E.M. *, P,0.05 vs. untreated transgenic hearts; †, P,0.05 vs. EHNA-treated transgenic hearts; ‡, P,0.05 transgenic vs. wild-type hearts. 4. Discussion The primary goal of the present study was to characterize cardioprotection due to adenosine metabolism inhibition in the ischemic–reperfused mouse heart. While inhibition of adenosine deaminase can prove beneficial during ischemia–reperfusion [5–15,31–34], mechanisms of protection remain unclear and there are presently no reports of the functional effects of adenosine kinase inhibition during myocardial ischemia–reperfusion. 126 J. Peart et al. / Cardiovascular Research 52 (2001) 120 – 129 enhanced adenosine receptor activation [1,2,22,26,27,36– 38] and (iii) reduced xanthine oxidase derived radical levels [3,4,14,15]. Since iodotubercidin blocks adenosine phosphorylation and enhances xanthine oxidase reactant levels, protection via this agent likely involves enhanced adenosine receptor activation. To test the roles of these different mechanisms, we studied the effects of co-infusion of iodotubercidin and EHNA, adenosine receptor antagonism and inosine and hypoxanthine infusion. We also compared responses to those for adenosine itself and examined effects of metabolism inhibitors in transgenic hearts overexpressing A 1 ARs. 4.2. Role of purine salvage in cardioprotection with EHNA and iodotubercidin Fig. 4. Post-ischemic LDH efflux from (A) wild-type and (B) transgenic mouse hearts subjected to 20 min global ischemia and 40 min reperfusion. Wild-type and transgenic hearts overexpressing A 1 ARs were either untreated (n514 and 8, respectively), or treated with 50 mM EHNA (n512 and 7, respectively), 10 mM iodotubercidin (n58 and 8, respectively), 50 mM EHNA110 mM iodotubercidin (n58 and 7, respectively), 50 mM 8-r-sulfophenyltheophylline (n58 wild-type hearts), 50 mM EHNA150 mM 8-r-sulfophenyltheophylline (n57 wild-type hearts), 10 mM iodotubercidin150 mM 8-r-sulfophenyltheophylline (n58), or 10 mM adenosine (n58). See key to Figs. 3 and 4 for abbreviations. Values are means6S.E.M. *, P,0.05 vs. untreated hearts; †, P,0.05 cotreatment vs. individual metabolism inhibitor treatment; ‡, P,0.05 transgenic vs. wild-type hearts. 4.1. Cardioprotection with EHNA and iodotubercidin Adenosine deaminase inhibition with EHNA significantly protected mouse hearts from ischemia–reperfusion, evidenced by reduced contractile dysfunction (Fig. 2) and enzyme loss (Fig. 4). Adenosine kinase inhibition with iodotubercidin provided a similar degree of protection (Figs. 2 and 4). Although a number of studies have assessed neuroprotective effects of adenosine kinase inhibition [16–18,35], these are the first data regarding the effects of adenosine kinase inhibition in ischemic–reperfused myocardium. Potential mechanisms involved in protection with EHNA include: (i) increased purine salvage [5,6,13]; (ii) Inhibition of adenosine kinase abolished cardioprotection via EHNA (Figs. 2 and 4), indicating that adenosine phosphorylation is essential for cardioprotection with the adenosine deaminase inhibitor. Similarly, addition of EHNA to iodotubercidin-treated hearts abolished cardioprotection. These data demonstrate that elevated extracellular adenosine at the expense of either adenosine phosphorylation or deamination is beneficial, whereas elevated adenosine at the expense of both paths simultaneously affords no protection. A path of adenosine salvage is therefore essential for full expression of the cardioprotective effects of adenosine catabolism inhibition. Adenosine can be reincorporated into the nucleotide pool via adenosine kinase (utilizing a single phosphate bond) or via conversion of hypoxanthine to IMP and then to 59-AMP (requiring two phosphates from 5-phosphoribose-1-pyrophosphate and GTP and resulting in ultimate hydrolysis of pyrophosphate formed). Since the activity of the former path is blocked by iodotubercidin and the latter is blocked by EHNA, coinfusion of both agents eliminates adenosine salvage and may deplete the nucleotide pool. As noted by Ryszard et al. [39], prolonged treatment of isolated myocytes with EHNA and iodotubercidin can deplete ATP, and this was only countered when ribose and adenine were supplied as alternate sources of purine moieties [39]. 4.3. Roles of receptor activation and catabolite formation Since 8-r-sulfophenyltheophylline reduced the effects of EHNA and iodotubercidin (Fig. 2), adenosine receptor activation appears to be involved in observed cardioprotection. This is consistent with the fact that overexpression of EHNA, iodotubercidin, adenosine and A 1 AR all confer almost identical protection from ischemia (Figs. 2–4). On the other hand, reduced xanthine oxidase derived radical formation, as a result of adenosine deaminase inhibition [3,4,14,15], does not appear to play a role. Iodotubercidin increases xanthine oxidase reactant levels (Table 2), as does adenosine infusion, yet both produce protection J. Peart et al. / Cardiovascular Research 52 (2001) 120 – 129 similar to that achieved with EHNA (Fig. 2). In addition, elevated intra-vascular inosine and hypoxanthine fails to alter post-ischemic recovery and also fails to reduce EHNA-mediated cardioprotection (see Results). Levels of inosine and hypoxanthine infused exceed those accumulating extracellularly during ischemia, as assessed via microdialysis [40]. No apparent relation seems to exists between functional recovery and xanthine oxidase reactant levels in the groups studied. Furthermore, 30–40% of ischemic / post-ischemic inosine and its catabolites are generated independently of adenosine deamination (Table 2), in agreement with data for the rat [25]. Since intra- and extracellular hypoxanthine and xanthine levels during ischemia–reperfusion may be 100-fold higher than the Km for xanthine oxidase [14,30,41,42], substrate maximally saturates the enzyme and a 60–70% reduction in substrate levels with EHNA treatment (based on 30–40% contribution from IMP) would not substantially reduce reaction rate during ischemia–early reperfusion, when xanthine oxidase is thought to play a role. This is consistent with the lack of effect of inosine and hypoxanthine infusion, and with previous observations that hypoxanthine and xanthine do not modify ischemic tolerance [43]. Since adenosine receptor activation reduces mitochondrial radical formation [44] and oxidant injury [36,37] and increases cardiovascular antioxidant capacity [38], correlations between radical levels and contractile recovery in EHNA treated hearts [14,15] likely reflect the beneficial effects of adenosine receptor activation and enhanced purine salvage via adenosine kinase rather than reduced xanthine oxidase derived radical formation. 4.4. Effects of adenosine metabolism inhibition in hearts overexpressing A1 ARs A 1 AR overexpression confers substantial receptor-mediated cardioprotection during ischemia–reperfusion [26,27,45], and this may involve mitochondrial KATP activation [27] and improved energy metabolism [26]. In the present study we tested whether inhibition of adenosine metabolism might confer added protection in these hearts. As shown in Figs. 2–4, A 1 AR overexpression, EHNA and iodotubercidin all reduce post-ischemic contractile dysfunction and enzyme efflux, and EHNA or iodotubercidin provide additional protection in transgenic hearts. Post-ischemic recovery in transgenic hearts treated with the inhibitors is remarkable, with function approaching that in pre-ischemic hearts (Fig. 3). These data indicate that A 1 AR-mediated protection in transgenic hearts is submaximal and enhanced by further elevations in adenosine, and / or that enhanced activation of other adenosine receptor subtypes (e.g. A 3 receptors) may be protective in this model. Since effects of preconditioning and A 1 AR overexpression are non-additive [45], these observations also indicate that EHNA and iodotubercidin must protect 127 via mechanisms in addition to those involved in preconditioning. 4.5. Selective effects of adenosine metabolism inhibition on ischemic contracture Ischemic contracture development, which is an indicator of severity of ischemic insult and damage [46], was selectively modified by the different treatments studied. Peak contracture was relatively insensitive to treatment whereas rapidity of contracture was variable (Fig. 1). This is consistent with the notion that peak contracture is related to the amount of ATP generated anaerobically and subsequently hydrolysed during ischemia [46]. It is interesting that EHNA was the most effective strategy in reducing contracture development (Fig. 1), surpassing the modest effects of adenosine and the insignificant reduction with iodotubercidin. These data imply differing mechanisms of action for these treatments. Minor effects of adenosine (and adenosine receptor antagonism) are consistent with our previous observations in murine heart [40] and the observations of Grover et al. in rat [47]. Lack of effect of iodotubercidin, and inhibition of the effects of EHNA by iodotubercidin indicate that reduced contracture development with elevated adenosine requires both adenosine phosphorylation and receptor activation. This is supported by paradoxically accelerated contracture in hearts treated simultaneously with iodotubercidin and 8-r-sulfophenyltheophylline (Fig. 1). 5. Conclusions In summary, our data reveal that inhibition of either adenosine deaminase or adenosine kinase elevates endogenous adenosine levels and significantly protects mouse hearts from ischemia–reperfusion. Cardioprotection due to EHNA is countered by inhibition of adenosine kinase, and protection with iodotubercidin is countered by simultaneous inhibition of adenosine deaminase. Thus, EHNA or iodotubercidin are only cardioprotective when a path of adenosine metabolism and salvage remains active. Inhibitory effects of adenosine receptor antagonism also reveal a key role for adenosine receptor activation. 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