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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.
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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. The present
results do not support a significant role for reduced
formation of reactants for xanthine oxidase in EHNAmediated cardioprotection. We also show that the protective effects of A 1 AR overexpression are submaximal, and
that augmentation of endogenous adenosine levels with
either EHNA or iodotubercidin further enhances recovery
in these hearts. Finally, we provide evidence that both
adenosine rephosphorylation and adenosine receptor activation are important in reducing contracture development
during ischemia.
128
J. Peart et al. / Cardiovascular Research 52 (2001) 120 – 129
Acknowledgements
This work was supported by grants from the National
Heart Foundation of Australia (G 98B 0080 and G 99B
0246) and a National Institutes of Health RO1 grant (HL
59419). The authors are extremely grateful for the excellent technical assistance of Bronwyn Garnham.
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