(Circulation. 1997;96:3094-3103.)
© 1997 American Heart Association, Inc.
Articles |
Importance of Endogenous Adenosine During Ischemia and Reperfusion in Neonatal Porcine Hearts
From the University of Oslo, Institute for Experimental Medical Research (H.T.S., F.G., J.O., A.I., K.A.K.) and Department of Anesthesia (K.A.K.), Ullevål Hospital, Oslo, and Institute for Physiology and Biomedical Techniques, Medical Technical Center, Trondheim (P.J.), Norway.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background Adenosine has several potentially cardioprotective effects. We hypothesized that the effects of endogenous adenosine vary with degree of ischemia and that elevating endogenous levels is protective.
Methods and Results Isolated blood-perfused piglet hearts underwent 120 minutes of low-flow ischemia (10% flow) or 90 minutes of zero-flow ischemia, all with 60 minutes of reperfusion. Hearts were treated with either saline, the adenosine receptor blocker 8-sulfophenyltheophylline (8SPT, 300 µmol · L-1), or the nucleoside transport inhibitor draflazine (1 µmol · L-1). In separate groups, biopsies were obtained before and at the end of ischemia. Compared with saline, 8SPT did not significantly alter functional recovery in either protocol. Draflazine significantly improved percent recovery of left ventricular systolic pressure both in the low-flow protocol (92±3% versus 75±2% [saline] and 73±3% [8SPT], P<.001 for both) and in the zero-flow protocol (76±3% versus 59±4% [saline] and 46±9% [8SPT], P<.05 for both). In the zero-flow protocol, draflazine also significantly reduced ischemic contracture and release of creatine kinase. Tissue adenosine at the end of ischemia was elevated by draflazine compared with saline-treated hearts: after low-flow ischemia to 0.10±0.05 versus 0.00±0.00 µmol · g-1 dry wt (P<.05) and after zero-flow ischemia to 1.73±0.82 versus 0.15±0.03 µmol · g-1 dry wt (P<.05).
Conclusions In neonatal porcine hearts, endogenous adenosine produced during ischemia does not influence ischemic injury or functional recovery. Elevating endogenous adenosine by draflazine elicits cardioprotection in both low-flow and zero-flow conditions.
Key Words: adenosine • infarction • ischemia • myocardium • reperfusion
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Adenosine has been shown to attenuate several deleterious effects of ischemia and reperfusion,1 and exogenous administration reduces infarct size,2 attenuates development of stunning,3 4 and mimics ischemic preconditioning.5 Thus, protective effects of exogenous adenosine are achieved, even though the endothelium serves as a barrier for its distribution.6 7
During ischemia, adenosine is endogenously produced because of breakdown of ATP.1 8 Endogenous adenosine has easy access to the interstitial space. However, a cardioprotective role is not intuitive, because adenosine is rapidly taken up by endothelial cells and catabolized or washed out.9 Conflicting data have been reported on the importance of endogenous adenosine produced during LF ischemia.10 11 12 13 14 15 During ZF ischemia, adenosine receptor inhibitors and adenosine deaminase impair functional recovery,16 17 whereas effects on infarct size are conflicting.18 19 20 21 22 Only one of these studies has been performed on neonatal hearts,13 which are more resistant to ischemia than adult hearts23 and differ in adenosine metabolism.24 25 26 The first aim of the present study was therefore to examine the role of endogenous adenosine produced during LF and ZF ischemia. This was achieved by selective blockade of adenosine A1 and A2 receptors with 8SPT.27
An alternative to exogenous administration of adenosine is to elevate levels of endogenous adenosine by inhibiting its degradation. After ZF ischemia, such interventions have been shown to reduce infarct size28 29 30 31 and improve functional recovery.32 33 34 However, negative findings have also been reported.35 36 37 None of these studies were performed in neonatal hearts. To the best of our knowledge, this approach has not previously been investigated in LF ischemia. There are profound differences between LF and ZF ischemia with regard to accumulation of metabolites and ions and formation of free oxygen radicals during reperfusion.38 Formation of adenosine is also related to the degree of ischemia.8 The second aim of the present study was therefore to examine whether elevated endogenous levels of adenosine can induce beneficial effects in LF and ZF conditions. We used draflazine, an inhibitor of the nucleoside transporter, which inhibits uptake of adenosine into endothelial cells and red blood cells and hence prevents intracellular degradation and transport across the endothelium.9 It is selective for the nucleoside transporter and devoid of other known effects.39
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Methods |
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Animals were maintained and housed in accordance with the conditions set by the Norwegian Council for Animal Research. The investigation 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 1985).
Heart Excision, Perfusion System, and Perfusate
Piglets were anesthetized with pentobarbital sodium (25
mg · kg-1 IP and 
25 mg ·
kg-1 IV) and given sodium heparin (1000 IU IV)
for anticoagulation. Mechanical ventilation through a tracheostoma was
performed with a mixture of 50% air/50% oxygen. Hearts were rapidly
excised and within seconds placed in 4°C KH buffer. A latex balloon
was passed into the left ventricle through the left atrium. Hearts were
mounted on the perfusion system by aortic cannulation within 10 minutes
after excision, and the pulmonary artery was cannulated. Hearts
were retrogradely perfused in a recirculating manner either at constant
pressure (70 mm Hg) or at controlled coronary flow with a
precisely calibrated pump (Cole Palmer Instruments). The chamber
enclosing the heart and other components of the perfusion system were
water-jacketed to maintain temperature at 37°C. Details of the system
have been described previously.13 40
Oxygenation was achieved by passing a mixture of 95%
O2/5% CO2 through the perfusate. The
perfusate was filtered by a transfusion filter (20 µm).
Hearts were perfused with erythrocyte-enriched KH buffer. Packed human
erythrocytes were washed twice with KH buffer (without BSA and
insulin). The modified KH solution contained glucose 11 mmol
· L-1, BSA (Sigma) 2%, free fatty acids
(from the BSA) 0.15 mmol · L-1,
insulin (Human Velosulin) 50 IU · L-1,
and ions from the following salts (in mmol ·
L-1): NaCl 118.5, NaHCO3 25.0, KCl
4.7, KH2PO4 1.2, MgSO4 1.2, and
CaCl2 2.4. Erythrocytes were then suspended in this
solution to provide a hematocrit of 0.20.41
Cardiac Performance Measurements
LV pressure was recorded by the fluid-filled balloon in the
left ventricle, connected to a Gould pressure transducer (model P23 Gb,
Gould Instruments). HR was derived from the pressure curves. PP was
measured through another Gould pressure transducer connected to a side
branch of the aortic cannula. Hemodynamic variables
were continuously recorded on an eight-channel thermal array
oscillographic recorder (model OR 2300, Yokogawa). When high
resolution of data was required, the output from this recorder was
sampled at 100 Hz. Signals were transformed by an analog-to-digital
converter for further computation. LV pressure-volume curves were
obtained by adding known fluid volumes into the balloon to increase
LVEDP from 0 to 15 mm Hg. The slope of the relation between
changes in LV volume and LV diastolic pressure was used to
estimate LV diastolic wall stiffness. During
ischemia, a rise in LVEDP of 4 mm Hg was defined as
ischemic contracture. If LVEDP exceeded 4 mm Hg, fluid
was removed from the balloon, after which LVEDP was maintained at
1 mm Hg.
Experimental Procedure and Protocols
Hearts were initially perfused at constant pressure (70
mm Hg), and the first venous effluent was discarded. After 20 to 30
minutes of stabilization, we switched to a flow-controlled mode (2
mL · min-1 ·
g-1 heart wt).13 40 Hearts were
paced via atrial electrodes (165 bpm). Criteria for entering the
study were peak LVSP10 before ischemia >95
mm Hg and pressure-controlled flow <3 mL ·
min-1 · g-1
heart wt. Coronary flow was reduced stepwise to 50%, 25%, and
10% of control value at 1-minute intervals. After LF ischemia,
flow was restored in the same steps. After ZF ischemia, flow
was gradually restored within 10 minutes to minimize reperfusion
injury. In LF experiments, pacing was stopped 1 minute after 10% flow
was established, and during reperfusion, pacing started 1 minute after
100% flow was reached. In ZF experiments, pacing was stopped just as
zero flow was reached, and pacing started between 5 and 10 minutes of
reperfusion, depending on arrhythmias. If detrimental
arrhythmias developed, hearts were defibrillated with
electrical pads for internal use (8 J). If conversion was rapidly
achieved, the experiment was continued.
Protocols, number of hearts, and time points of acquisition of data and
blood samples are shown in Fig 1
. Hearts
were subjected to two different protocols, LF or ZF ischemia,
and exposed to saline, 8SPT, or draflazine from 20 minutes before
ischemia. Fifty-one piglets were randomized to six groups
subjected to LF or ZF ischemia with subsequent reperfusion to
obtain functional data and biopsies at the end of reperfusion (part A).
Forty-eight piglets were randomized to nine groups in which biopsies
were obtained before ischemia (C2) or at end of LF or ZF
ischemia to obtain biopsies at these times (part B). At points
of data acquisition, perfusate samples were obtained just
before recording of LV function, PP, and HR.
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, myocardial biopsies; 
, blood
samples and hemodynamic recordings;
Metabolic Measurements
Perfusate samples were obtained
anaerobically and simultaneously from the
pulmonary artery (venous samples) and from a side branch of the
aortic cannula (arterial samples). Lactate concentrations
were analyzed enzymatically by
spectrophotometry.42 Hemoglobin (Hb) was determined in
duplicate by the cyanomethemoglobin method. Arterial and
venous oxygen saturations (SaO2 and
SvO2) were analyzed on an IL 282
CO-Oximeter (Instrumentation Laboratories). Arterial and
venous partial pressures of oxygen (PaO2 and
PvO2) and partial pressures of carbon dioxide
(PaCO2 and PvCO2) and
pH were analyzed on an automatic blood gas analyzer
(model 945, AVL Biochemical Instruments). Myocardial oxygen consumption
(M
O2) was estimated by the formula
MO2 (µmol ·
min-1 · 100
g-1)=[(SaO2-SvO2)x62.1xHb/100+(PaO2-PvO2)x0.00141]xCBFx100/MW,
where SO2 is oxygen saturation in %,
PO2 is partial pressure of oxygen in
mm Hg, the constant 62.1 is oxygen binding capacity of Hb in
µmol · g-1, Hb is concentration of
hemoglobin in g · mL-1, the constant
0.00141 is the solubility constant of oxygen in blood per atmosphere at
37°C in µmol · mL-1
· mm Hg-1, CBF is coronary
blood flow in mL · min-1, and MW is
myocardial wet weight in g.
Tissue biopsies were obtained by freeze-clamping a part of the LV free wall and thereafter, they were freeze-dried within 24 hours and stored at -70°C before analysis. Biopsies obtained at the end of reperfusion were analyzed enzymatically by luminometry43 and at the end of ischemia by high-performance liquid chromatography.44 Enzyme activity of CK, in IU · L-1, was analyzed enzymatically by fluorescence in arterial and venous perfusate before ischemia and every 15 minutes during reperfusion. Release of CK was calculated by summation of the AV differences and presented as cumulative release. Water content, indicative of cellular edema, was expressed as percent of wet weight. Samples from the left ventricle were blotted to remove excess fluid and dried to constant weight at 37°C.
Pharmacological Agents
All agents were prepared the day of the experiment and
circulated for 20 minutes before ischemia. 8SPT (Research
Biochemicals Inc, catalog number A013) was dissolved in saline and
added to obtain a perfusate concentration of 300
µmol · L-1. It is a water-soluble
xanthine derivative that selectively blocks adenosine
A1 and A2 receptors,27 relatively
equipotent for the two subclasses.
Draflazine was kindly provided by H. Van Belle, Janssen Pharmaceutical, Beerse, Belgium. The agent was dissolved in saline and added to obtain a perfusate concentration of 1 µmol · L-1. It was carefully shielded from exposure to light during storage, preparation, and administration, and the perfusion system was wrapped in cellophane. Draflazine is the active (-)-enantiomer of the nucleoside transporter inhibitor R75231 and is highly potent and selective toward the transporter molecule.9 39 45
Statistical Analysis
Data are presented as mean±SEM. Regression lines
between LVSP and LVEDP were calculated, and values for LV function are
presented for LVEDP=10 mm Hg (LVSP10). A
paired t test was used to assess differences within groups
between C1 and C2. The Fisher exact test was used for comparison of
proportions. One-way ANOVA, followed by the Student-Newman-Keuls test,
was used for analysis of repeated measurements within groups
and for comparison of variables between three groups at single time
points. MANOVA, followed by the Student-Newman-Keuls test, was used for
multiple group comparisons of repeated measurements. A value of
P<.05 was considered statistically significant. Power
analyses (LVSP10 and CK) are described in the
"Results."
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Results |
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A total of 99 piglets were included in the study. In part A, 51 hearts were subjected to either LF or ZF ischemia, followed by 60 minutes of reperfusion. In part B, biopsies were obtained before (C2) or at the end of ischemia from 48 hearts. Pig weight, heart weight, age, and dose of pentobarbital sodium did not differ between groups (Table 1
). In part A, 14
piglets were excluded (3 in the LF and 11 in the ZF protocol) because
of technical problems (4), hemolysis of red blood cells (2), air emboli
(3), and unsuccessful defibrillation at reperfusion (5). In part B, 3
piglets were excluded (1 in the LF protocol and 2 in the C2 protocol)
because of technical problems (2) and ventricular
fibrillation (1). Data are presented from the remaining 82
piglets.
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Hemodynamic Effects of 8SPT and Draflazine
In control hearts, there was a significant fall in sHR and PP from
C1 to C2. In 8SPT-treated hearts, no changes were observed. In
draflazine-treated hearts, the decline in sHR and PP was accentuated.
In addition, LVSP and MO2 showed a
slight decline. Right before ischemia (C2), PP in
draflazine-treated hearts was reduced compared with the other
groups.
Mechanical Function and Metabolism During LF
Ischemia
Flow reduction to 10% reduced LVSP10, sHR, and
MO2 in all groups. There was no
significant difference in LVSP10 between groups during
ischemia (Fig 2). During the
first 90 minutes of ischemia, sHR and
MO2 were similar in hearts treated with
8SPT or saline (Fig 2, Table 2), but at
end of ischemia both variables were significantly higher in
8SPT-treated hearts (for both; P<.05). During the whole
ischemic period, sHR and MO2
were lower in draflazine-treated hearts than in both other groups. Only
minor changes in diastolic wall stiffness were found, and
none of the hearts developed ischemic contracture.
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), 8SPT (
), or draflazine (
P<.01, and
P<.001 for draflazine vs both saline and 8SPT.
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Arterial pH, PO2, and
SO2 were stable during the experimental period.
Arterial PCO2 and lactate increased
gradually over time, with no differences between groups. During
ischemia, all hearts showed signs of anaerobic
metabolism (Table 2
). During the first 90 minutes, AV
differences for pH, PCO2, and lactate were
similar in hearts treated with saline or 8SPT, but at the end of
ischemia, AV differences for pH and
PCO2 were significantly higher in 8SPT-treated
hearts. In draflazine-treated hearts, there were smaller changes in AV
differences for pH and PCO2 during the first 90
minutes of ischemia than in hearts treated with saline or 8SPT.
A similar pattern was observed for lactate, but it did not reach
statistical significance. At the end of ischemia, AV
differences in pH and PCO2 were similar in
hearts treated with saline or draflazine.
Mechanical Function During ZF Ischemia
After a complete stop of perfusion, hearts continued to beat
vaguely for some minutes. Ischemic contracture developed in
hearts treated with saline (8 of 10) and 8SPT (8 of 9), but not in
draflazine-treated hearts (0 of 8) (for draflazine versus both other
groups, P<.01). Diastolic stiffness increased
in all groups during ischemia, but this increase was lower in
draflazine-treated hearts (Fig 3).
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Mechanical Function and Metabolism During Reperfusion
After LF Ischemia
During reperfusion, there was immediate recovery in mechanical
function (Fig 2). Recovery of contractile function was not
significantly different in hearts treated with saline compared with
hearts treated with 8SPT at any time during reperfusion (see later
power analysis). Hearts treated with draflazine showed
significantly better recovery of function than the two other groups,
with LVSP10 after 60 minutes of reperfusion 92±3% of
preischemic value compared with 75±2% in saline-treated
hearts (P<.001) and 73±3% in 8SPT-treated hearts
(P<.001). During reperfusion, indices of
anaerobic metabolism ceased and AV differences
in pH, PCO2, and lactate returned to
preischemic values in all groups (Table 2).
Consistent with good functional recovery,
MO2 returned to almost
preischemic levels. Only in 8SPT-treated hearts was
MO2 slightly reduced compared with
preischemic values (P<.01).
Mechanical Function and Metabolism During Reperfusion
After ZF Ischemia
There was gradual improvement in systolic function during
the first 30 minutes of reperfusion, with subsequent stabilization (Fig 4). Hearts treated with 8SPT showed a
tendency toward reduced recovery compared with saline-treated hearts,
but this did not reach statistical significance at any time (see power
analysis). Draflazine-treated hearts showed better functional
recovery than the two other groups, with LVSP10 after 60
minutes of reperfusion 76±3% of preischemic value
compared with 59±4% in saline-treated hearts (P<.05) and
46±9% in 8SPT-treated hearts (P<.05).
Diastolic stiffness declined during reperfusion in
saline-treated hearts but remained slightly elevated compared with
preischemia (Fig 3). In 8SPT-treated hearts, stiffness
transiently increased during reperfusion and remained elevated compared
with preischemia. The slight increase in stiffness in
draflazine-treated hearts was fully reversed during reperfusion.
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Arterial pH, PO2, and
SO2 were stable during the experimental period.
Arterial PCO2 and lactate increased
gradually over time, with no differences between groups.
Consistent with incomplete functional recovery,
MO2 was significantly reduced in all
groups, with no group differences. At 30 minutes of reperfusion, hearts
treated with draflazine showed significantly smaller AV differences in
pH and PCO2 compared with hearts treated with
saline or 8SPT (Table 3). The same
tendency was observed for lactate, but it did not reach statistical
significance. At 60 minutes of reperfusion, all AV differences had
returned to preischemic levels.
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Release of CK During Reperfusion
After LF ischemia, release of CK (IU · 100
g-1) was low: 11±2 (saline), 8±2 (8SPT), and
8±3 (draflazine). There were no differences between groups. After ZF
ischemia, release of CK was higher: 140±3 (saline), 213±77
(8SPT), and 20±6 (draflazine). Draflazine-treated hearts showed
significantly lower release (versus both other groups,
P<.05), whereas there was no observed difference between
hearts treated with saline or 8SPT (see later power
analysis).
Tissue Metabolites and Water Content
Values obtained before (C2) and at the end of ischemia are
given in Table 4. Before
ischemia, there were no differences between groups in either
ATP, PCr, or adenosine. In the LF protocol, there were no
changes in ATP or PCr either during ischemia or during
reperfusion and no differences between groups. At the end of ZF
ischemia, ATP and PCr were significantly lower than before
ischemia in all groups and significantly lower in hearts
treated with 8SPT compared with the two other groups. At the end of
reperfusion after ZF ischemia, levels of ATP and PCr were
significantly higher in draflazine-treated hearts than in both other
groups (for both, P<.05). At the end of both LF
ischemia and ZF ischemia, levels of adenosine
were significantly increased in hearts treated with draflazine compared
with hearts treated with saline or 8SPT. In addition,
adenosine/inosine ratios were substantially elevated in
draflazine-treated hearts in both protocols (Table 4). At the end of
reperfusion, adenosine levels were still higher in
draflazine-treated hearts in the ZF protocol, but not in the LF
protocol. There were no differences in H2O% between groups
in either of the protocols (Table 4).
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Analysis of Power
A statistical analysis was performed to measure the
probability that true differences in functional recovery and CK release
in hearts treated with 8SPT compared with hearts treated with saline
were missed because of insufficient sample size (type II error). All
power calculations were made at 
=.05.
We calculated the mean of percent recovery of LVSP10 during the stable phase of reperfusion in all groups. The differences between groups in mean recovery in the LF protocol were 16% for saline- versus draflazine-treated hearts (P<.01), 17% for 8SPT- versus draflazine-treated hearts (P<.01), and 2% for 8SPT- versus saline-treated hearts (P=.73). Corresponding values in the ZF protocol were 21% (P<.05), 32% (P<.01), and 12% (P=.19). In the LF protocol, the probability of detecting a 30% alteration in 8SPT-treated hearts compared with saline-treated hearts was 99%. In the ZF protocol, the probability of detecting a 30% alteration in 8SPT-treated hearts compared with saline-treated hearts was 47%. To achieve an 80% probability of detecting this difference, we would have needed 20 animals in each group.
Power calculations were also done for CK release in the ZF protocol. The differences between groups in mean cumulative CK release (IU · 100 g-1) were 120 for saline- versus draflazine-treated hearts, 193 for 8SPT- versus draflazine-treated hearts (for both, P<.05), and 73 for 8SPT- versus saline-treated hearts (P=.29). Draflazine treatment caused an 86% decrease in CK release compared with saline-treated hearts. To achieve an 80% probability of detecting a 50% alteration in 8SPT-treated hearts compared with saline-treated hearts, we would have needed 74 animals in each group.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The role of endogenous adenosine was investigated during LF and ZF ischemia and reperfusion in neonatal porcine hearts. Blockade of adenosine receptors did not significantly affect functional recovery in either condition. Elevating adenosine levels by the nucleoside transporter inhibitor draflazine improved functional recovery after both LF and ZF ischemia. Also, in ZF conditions, ischemic contracture and CK release were attenuated.
Methodological Considerations
The isolated heart preparation is a well-established model for
studying metabolism and function during both LF and ZF
ischemia. Confounding systemic reactions triggered in the
intact organism are eliminated, including neurohumoral mechanisms and
alterations in plasma levels of bioamines, hormones, and substrates.
This allows investigation of intrinsic functional and
metabolic properties of the heart in response to
ischemia. The erythrocyte-enriched perfusate provides
adequate oxygenation at
physiological flow rates and enables us to closely
mimic LF ischemia, in which rate of washout of metabolites and
ions is of crucial importance. This is in sharp contrast to
crystalloid-perfused preparations, in which coronary flow has
to be 5 to 10 times above normal to ensure adequate
oxygenation. In our preparation, we have previously
confirmed stable mechanical and metabolic function for at
least 3 hours.13 46
In global ischemia, at least two determinants of energy consumption are substantially reduced. First, passive repetitive stretching of the ischemic myocardium is an important determinant for metabolism,46 and second, HR is severely reduced. Also, neonatal hearts tolerate ischemia better than adult hearts.23 Developmental differences in calcium regulation, glycogen content, and enzyme activities in metabolic pathways may contribute to our findings.23 Eventually, the isolation procedure could alter baseline contractile function and responses to ischemia, but all groups would be equally affected.
Role of Endogenous Adenosine Produced
During Ischemia
The importance of endogenous adenosine
produced during ischemia was investigated by blocking
adenosine receptors with 8SPT. This did not affect functional
recovery after either LF or ZF ischemia or CK release after ZF
ischemia, indicating that the amount of adenosine
produced during ischemia is too low to have
physiological effects on functional recovery. Our
data on LF ischemia are conclusive (see power analysis)
and in accordance with previous studies in both
isolated13 14 15 and intact hearts.10 In ZF
conditions, we cannot rule out that there was a small effect of 8SPT. A
tendency toward impaired recovery and increased release of CK was
observed, but it did not reach statistical significance. However, we
would have needed prohibitively large sample sizes to detect these
differences (see power analysis). In ZF ischemia, some
studies have shown no effect of 8SPT on infarct size,18 19 20
whereas others have found increased infarction.21 22
During early LF ischemia, there were no differences in
mechanical function and metabolism between hearts treated
with saline or 8SPT. This is in accordance with previous studies in
intact animals10 and neonatal13 and
adult15 isolated hearts, in which endogenous
adenosine does not affect initial downregulation of function.
However, toward the end of ischemia, hearts treated with 8SPT
showed increased sHR, MO2, and AV
differences for pH and PCO2 and similar
tendencies for lactate and LVSP10. These alterations are
most likely due to blockade of A1 receptors, which results
in increased HR and inotropy. Whereas two studies have shown that
antagonism of adenosine with 8SPT13 or
adenosine deaminase10 during LF ischemia
does not affect metabolism, another study has reported
increased release of lactate.14 Discrepancies might be due
to degree and duration of flow reduction and the species and age of
animals used.
The prominent increase in diastolic stiffness in 8SPT-treated hearts during ZF ischemia is in accordance with earlier works.47 48 49 In 8SPT-treated hearts, we found that diastolic stiffness was transiently further increased during early reperfusion, whereas in saline-treated hearts, stiffness was partially reversed. Also, we found no relation between ATP and PCr levels at the end of ischemia and contracture development during ischemia. Hence, it is likely that contracture development is not only due to a decline in ATP but also may involve other factors, such as elevation in intracellular calcium ions.50 Levels of ATP and PCr at the end of ZF ischemia were significantly lower in hearts treated with 8SPT than in saline-treated hearts. Thus, endogenous adenosine preserves cellular energy stores during severe ischemia. However, we found no relation between total tissue ATP and PCr at the end of ischemia and recovery of function.
The agent 8SPT blocks adenosine A1 and A2 receptors relatively equipotently.27 Because of the polar sulfophenyl group, 8SPT does not cross the myocyte plasma membrane. Hence, it does not inhibit cAMP phosphodiesterase activity or interfere with release of adenosine from myocytes. We have previously examined the dose-response characteristics and duration of 8SPT in this preparation and shown that the concentration used gives complete blockade of adenosine receptors throughout the experimental protocol.13 Thus, lack of effect of 8SPT is not due to insufficient blockade of adenosine receptors. The use of neonatal hearts can be of importance. These hearts have greater intrinsic glycolytic capacity,51 and release of adenosine might be lower because of lower activity of the 5'-nucleotidase25 26 and preferential deamination of AMP to IMP instead of dephosphorylation to form adenosine.24
Effect of Elevating Endogenous Levels of
Adenosine: Possible Mechanisms of Action
Even though endogenous adenosine produced
during ischemia does not seem to affect functional recovery, we
show that elevating endogenous adenosine levels by
draflazine improves functional recovery after both LF and ZF
ischemia. This indicates that nonprotective levels of
adenosine can be augmented to elicit protection. To the best of
our knowledge, the present study is the first to show that
elevating endogenous adenosine is protective in LF
conditions as well. This is not obvious because there are great
differences between LF and ZF ischemia with regard to
accumulation of metabolites and ions and formation of free oxygen
radicals upon reperfusion.38 Also, the amount of
adenosine produced during ischemia depends on the
degree of reduction in flow.8 Improved functional recovery
after ZF ischemia has been shown after treatment with
nucleoside transport inhibitors or inhibitors
of adenosine deaminase during
ischemia,32 33 34 52 53 54 55 56 57 but not in one study in
isolated whole-blood–perfused rabbit hearts.37
Release of CK after ZF ischemia was reduced after treatment with draflazine. Reduction in infarct size has previously been shown after treatment with the nucleoside transporter inhibitors R7523128 29 and S-(p-nitrobenzyl)-6-thioinosine30 and inhibitors of adenosine deaminase,31 with one exception.36 To the best of our knowledge, the approach of elevating endogenous levels of adenosine has not previously been tested in neonatal hearts, but it has been shown to be protective against cerebral infarction in newborn rats.58
Even though endogenous adenosine is not responsible
for downregulation of mechanical function during LF
ischemia,10 11 12 13 14 15 our results show that when
endogenous adenosine is augmented, mechanical
function is further reduced. The negative chronotropic and inotropic
effects are probably mediated by A1 receptors, which also
antagonize ß-adrenergic actions.59 In parallel with
reduced mechanical function during ischemia, draflazine-treated
hearts had reduced MO2 and reduced signs
of anaerobic metabolism. Draflazine is a
nucleoside transport inhibitor and binds with high affinity
to the nucleoside transporter in cell membranes of
endothelial cells, erythrocytes, and pericytes. This
inhibits cellular uptake of adenosine and other nucleosides and
prevents intracellular degradation and transport across the
endothelium.9 Thus, endogenous
adenosine accumulates in the interstitial space,
and the intravascular level also increases.9 Unlike
dipyridamole, it does not affect the phosphodiesterase
activity or prostacyclin formation. Biopsies at the end of LF and ZF
ischemia confirmed higher levels of adenosine in
draflazine-treated hearts than in both other groups. Because draflazine
has been shown to be devoid of effects other than nucleoside transport
inhibition both in vivo and in vitro,39 its effects are
due to increased myocardial adenosine levels.
Adenosine can exert protective effects during ischemia as well as during reperfusion. Activation of A1 receptors on the myocyte seems most important during ischemia,1 mediating negative chronotropic and inotropic effects. During reperfusion, A2-mediated vasodilatation and inhibition of activated blood cells seem to be most important.1 In our study, these actions are probably of minor importance. Lack of blood elements other than erythrocytes eliminates effects on neutrophils and platelets. Flow-controlled perfusion reduces the importance of vasodilatation, although an effect on distribution might exist during LF ischemia and during reperfusion. Nucleoside transport inhibition leads to reduced formation of substrates for xanthine oxidase, which will reduce formation of harmful free oxygen radicals during reperfusion.57 This effect is also of minor importance, because porcine myocardium, like human myocardium, has no detectable xanthine oxidase.60 Other protective effects of elevated adenosine levels might involve reduced lipid peroxidation, reduced damage by free oxygen radicals,1 or altered glucose metabolism.15 49 50 61
We observed almost no increase in diastolic stiffness during ZF ischemia in draflazine-treated hearts. Because draflazine also altered function and metabolism during LF ischemia, this indicates that the protective effects of elevated adenosine levels are at least partly exerted during ischemia. A1 receptor activation may inhibit calcium overload50 through opening of ATP-sensitive potassium channels62 and also antagonize adrenergic effects.59 These mechanisms might contribute to the reduced ischemic contracture in draflazine-treated hearts. At the end of ZF ischemia, the declines in both ATP and PCr were similar in control hearts and hearts treated with draflazine. However, at the end of reperfusion after ZF ischemia, draflazine-treated hearts showed significantly higher levels of both metabolites compared with control hearts. Thus, elevated endogenous adenosine during severe ischemia seems to have beneficial effects both on recovery of function and generation of high-energy phosphates during reperfusion.63
Conclusions
Our results show that endogenous adenosine
produced during LF and ZF ischemia does not significantly
affect functional recovery or cell necrosis in neonatal porcine hearts.
However, elevating adenosine levels by the nucleoside transport
inhibitor draflazine improves functional recovery and
prevents cell necrosis. This indicates that nonprotective levels of
adenosine can be augmented to elicit protection if degradation
of adenosine is inhibited. In treatment of patients with acute
myocardial ischemia, increased endogenous
adenosine levels may extend the time window for successful
reperfusion. This approach provides a time- and site-specific therapy
that minimizes unwanted systemic effects.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the University of Oslo, Anders Jahre's Fund for Promotion of Science, Professor Carl Semb's Medical Research Fund, The Lærdal Foundation for Acute Medicine, and The Norwegian Air Ambulance Foundation. The authors acknowledge the technical support of Hilde Dishington, Unni Henriksen, Mette Ree Holte, Severin Leraand, Thea Sandsbråthen, Sissel Skarra, and Turid Verpe. Analysis of CK was kindly offered by Petter Urdal at Department of Clinical Chemistry, Ullevål Hospital. Special thanks to Morten Eriksen for skilled assistance throughout the experiments.
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Footnotes |
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Reprint requests to Dr Hilchen T. Sommerschild, Institute for Experimental Medical Research, Ullevål Hospital, N-0407 Oslo, Norway.
Received February 26, 1997; revision received June 3, 1997; accepted June 14, 1997.
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References |
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