(Circulation. 1996;93:1725-1733.)
© 1996 American Heart Association, Inc.
Articles |
Dichotomy of Ischemic Preconditioning
Improved Postischemic Contractile Function Despite Intensification of Ischemic Contracture
From Cardiovascular Research, the Rayne Institute, St Thomas' Hospital, London, UK.
Correspondence to K.G. Kolocassides, Cardiovascular Research, Rayne Institute, St Thomas' Hospital, London SE1 7EH, UK.
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Abstract |
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Background Acceleration of ischemic contracture is conventionally accepted as a predictor of poor postischemic function. Hence, protective interventions such as cardioplegia delay ischemic contracture and improve postischemic contractile recovery. We compared the effect of ischemic preconditioning and cardioplegia (alone and in combination) on ischemic contracture and postischemic contractile recovery.
Methods and Results Isolated rat hearts were aerobically perfused with blood for 20 minutes before being subjected to zero-flow normothermic global ischemia for 35 minutes and reperfusion for 40 minutes. Hearts were perfused at a constant pressure of 60 mm Hg and were paced at 360 beats per minute. Left ventricular developed pressure and ischemic contracture were assessed with an intraventricular balloon. Four groups (n=8 hearts per group) were studied: control hearts with 35 minutes of unprotected ischemia, hearts preconditioned with one cycle of 3 minutes of ischemia plus 3 minutes of reperfusion before 35 minutes of ischemia, hearts subjected to cardioplegia with St Thomas' solution infused for 1 minute before 35 minutes of ischemia, and hearts subjected to preconditioning plus cardioplegia before 35 minutes of ischemia. After 40 minutes of reperfusion, each intervention produced a similar improvement in postischemic left ventricular developed pressure (expressed as a percentage of its preischemic value: preconditioning, 44±2%; cardioplegia, 53±3%; preconditioning plus cardioplegia, 54±4%; and control, 26±6%, P<.05). However, preconditioning accelerated whereas cardioplegia delayed ischemic contracture; preconditioning plus cardioplegia gave an intermediate result. Thus, times to 75% contracture were as follows: control, 14.3±0.4 minutes; preconditioning, 6.2±0.3 minutes; cardioplegia, 23.9±0.8 minutes; and preconditioning plus cardioplegia, 15.4±2.4 minutes (P<.05 preconditioning and cardioplegia versus control). In additional experiments, using blood- and crystalloid-perfused hearts, we describe the relationship between the number of preconditioning cycles and ischemic contracture.
Conclusions Although preconditioning accelerates, cardioplegia delays, and preconditioning plus cardioplegia has little effect on ischemic contracture, each affords similar protection of postischemic contractile function. These results question the utility of ischemic contracture as a predictor of the protective efficacy of anti-ischemic interventions. They also suggest that preconditioning and cardioplegia may act through very different mechanisms.
Key Words: ischemia • phosphates • metabolism • prevention • cardioplegia
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Introduction |
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Within seconds of the onset of myocardial ischemia, hearts exhibit acute contractile failure, which may result in complete diastolic arrest within 2 to 3 minutes. The contractile apparatus then forms new cross bridges with the gradual development of tension. This process is known as ischemic contracture; in its extreme form, this presents clinically as the "stone heart,"1 which was thought to be an irreversible condition that contributed to intraoperative mortality.
When the introduction of myocardial protection combated ischemic injury,2 it was realized that interventions that reduced postischemic contractile dysfunction also delayed the progress of ischemic contracture.3 At that time, various histological and functional studies associated the onset of ischemic contracture with the onset of irreversible cell damage.4 The link between contracture and ischemic injury was so strong that it became acceptable to use the timing of ischemic contracture as a reliable index with which to compare the anti-ischemic efficacy of various interventions5 6 and to compare the susceptibility to ischemic injury of hearts from animals of different species7 or ages.8 Only a few studies have questioned either the association between ischemic contracture and irreversible injury or the value of ischemic contracture as a predictor of postischemic recovery.9 In neonatal hearts,10 11 there was no correlation between contracture, ischemic injury, and postischemic functional recovery.
Preconditioning has been described as one of the most powerful means of protecting the ischemic myocardium,12 with an ability to limit infarct size, improve postischemic contractile function, and reduce the severity of ischemia- and reperfusion-induced arrhythmia.12 The literature on ischemic preconditioning contains relatively few observations to suggest that ischemic preconditioning might not reduce the severity of ischemic contracture13 and might exacerbate it.14 15
The objectives of the present study were to use the blood- and crystalloid-perfused rat heart to examine whether ischemic preconditioning exacerbates ischemic contracture; the perfusion medium and the "dose" of preconditioning influence this phenomenon, together with changes in glycolytic metabolism; the accelerated contracture associated with ischemic preconditioning attenuates the protection of contractile function; and measures to reduce ischemic contracture might increase the protection of contractile recovery afforded by preconditioning.
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Methods |
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Animals
Male Wistar rats (Bantin and Kingman) were used both as donor (250±10 g body wt) and support (400±5 g body wt) animals. All rats received proper care in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the NIH (NIH publication No. 85-23, revised 1985). All rats were anesthetized with pentobarbital (60 mg/kg body wt IP) and then heparinized with heparin (1000 IU/kg body wt IV).
Blood-Perfused Heart Preparation
Support Rats
A Langendorff circuit was supplied with blood from rats placed
on femoro-femoral bypass by open cannulation of the right femoral
artery and left femoral vein. The extracorporeal circuit was primed
with 3.5% colloid (Haemaccel; 3.75 mL/100 g body wt of the support
rat), and closed circulation was established for 20 minutes to ensure
good mixing with the blood of the support rat. The outflow from the
femoral artery was controlled by a calibrated roller pump. The venous
return was gravitational and controlled by a gate clamp. The support
rats lay supine on a thermostatically controlled surface set to
maintain a body temperature of 36°C to 37°C. Although
self-ventilating, a mixture of 95% oxygen and 5% carbon dioxide
was supplied through a 24% Venturi mask placed over the face of the
rat. An arterial PO2 of 160 to 180
mm Hg was achieved. Anesthesia was maintained with
pentobarbital administered as a bolus (3 to 6 mg) to the venous return
reservoir. The respiratory rate was kept between 60 and 80 breaths per
minute to maintain the arterial
PCO2 in the physiological
range (40 to 60 mm Hg).
Isolated Hearts
Hearts were rapidly excised from anesthetized rats and
immersed in cold (4°C) Ringer's solution. The aorta was cannulated
onto a stainless steel cannula dripping arterial blood. The
pulmonary artery was incised. Atrial pacing was established at
360 beats per minute, and blood flow was adjusted to maintain a
constant perfusion pressure of 60 mm Hg. A left
intraventricular balloon was introduced through the
mitral valve for measurement of left ventricular developed
pressure (LVDP). The balloon was inflated with water to maintain a
constant left ventricular end-diastolic
pressure (LVEDP) of 4 mm Hg.
Hearts were aerobically perfused at 35.5±0.1°C16 for 20 minutes; then preischemic LVDP was recorded. Zero-flow global ischemia was induced by clamping of the arterial line. During ischemia, the hearts were maintained at 37°C (without pacing) by immersion in a thermostatically controlled chamber containing Ringer's solution. Reperfusion was gradually restored over 1 minute, and perfusion pressure again was set at 60 mm Hg. The intraventricular balloon also was deflated sufficiently to reestablish an LVEDP of 4 mm Hg. After 40 minutes of reperfusion, LVDP was again recorded.
Crystalloid-Perfused Heart Preparation
Hearts were perfused in the Langendorff mode by use of a
bicarbonate buffer at 36.5°C and a constant perfusion pressure of 73
mm Hg. All other components of the system were the same as for the
blood-perfused preparation.
Solutions
Bicarbonate buffer ([mmol/L] NaCl 118.5, KCl 5.9,
CaCl2 1.4, NaHCO3 25.0, MgSO4 1.2,
and glucose 11.0) was gassed with 95% O2 plus 5%
CO2 (PO2 of 550 mm Hg, pH 7.4). The
St. Thomas' cardioplegic solution No. 2 ([mmol/L] NaCl 110.0,
KCl 16.0, MgCl2 16.0, CaCl2 1.2, and
NaHCO3 10.0) was titrated to pH 7.8 and filtered. The
Haemaccel contained (mmol/L) Na 145.0, K 5.1, Ca 6.3, and Cl 145.0 and
trace amounts of inorganic phosphate, sulfate, and gelatin (molecular
weight, 35 000; 35 g/L). The Ringer's solution contained (mmol/L) Na
147.0, K 4.0, Ca 2.2, and Cl 156.0.
Experimental Protocols
Study 1: Effect of Preconditioning and/or Cardioplegia on
Ischemic Contracture and Postischemic
Contractile Recovery
Fig 1
is a diagram of the protocols used. We used
blood-perfused hearts in four groups (n=8 per group): (1) control
hearts with unprotected ischemia, (2) hearts preconditioned
with one cycle of 3 minutes of ischemia plus 3 minutes of
reperfusion, (3) hearts treated with St Thomas' cardioplegic
solution infused at 37°C and 60 mm Hg for 1 minute, and (4) hearts
subjected to preconditioning plus cardioplegia before 35 minutes of
ischemia and 40 minutes of reperfusion.
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Study 2: Effect of the Extent of Development of Ischemic
Contracture on Postischemic Contractile
Recovery
Hearts were reperfused at a time when control and preconditioned
hearts exhibited markedly different degrees of ischemic
contracture. We used blood-perfused hearts in two groups (n=8 per
group): control hearts with unprotected ischemia and hearts
preconditioned with one cycle of 3 minutes of ischemia plus 3
minutes of reperfusion before ischemia. The ischemic
period was 7.5 minutes, a duration selected on the basis of the results
of study 1. For the metabolite analysis, additional hearts (n=4
per group) were frozen with liquid nitrogen–cooled tongs just
before and at the end of the 7.5-minute ischemic period. All
hearts were randomized together.
Study 3: Relationship Between the Number of Cycles of
Preconditioning and the Occurrence of Ischemic
Contracture
To compare our findings with other studies in the literature,
both blood- and crystalloid-perfused hearts were used as follows
(n=6 per group): A, blood-perfused hearts in four groups: (1)
control hearts with unprotected ischemia; (2), (3), and (4),
hearts preconditioned with 1, 2, and 3 cycles, respectively, of 3
minutes of ischemia plus 3 minutes of reperfusion before 20
minutes of ischemia; B, crystalloid-perfused hearts in four
groups: (1), control hearts with unprotected ischemia; (2),
(3), and (4), hearts preconditioned with 1, 2, and 3 cycles,
respectively, of 3 minutes of ischemia plus 3 minutes of
reperfusion before 20 minutes of ischemia; and C,
crystalloid-perfused hearts in two groups: (1), hearts subjected to
two cycles of preconditioning, the first with 3 minutes of
ischemia plus 3 minutes of reperfusion and the second with 5
minutes of ischemia plus 3 minutes of reperfusion; and (2),
hearts subjected to three cycles of preconditioning, the first with 3
minutes of ischemia plus 3 minutes of reperfusion, and the
second and third each with 5 minutes of ischemia plus 3 minutes
of reperfusion before 20 minutes of ischemia. In this study,
the groups in B and C were randomized together; the hearts were not
reperfused.
Study 4: Relationship Between the Number of Cycles of
Preconditioning and the Extent of Postischemic
Contractile Recovery
We used crystalloid-perfused hearts in three groups (n=6 per
group): (1) control hearts with unprotected ischemia, (2)
hearts preconditioned with one cycle of 3 minutes of ischemia
plus 3 minutes of reperfusion, and (3) hearts preconditioned with three
cycles of 3 minutes of ischemia plus 3 minutes of reperfusion
before 35 minutes of ischemia and 40 minutes of
reperfusion.
Exclusion Criteria
Blood-perfused hearts that after the first 20 minutes of
aerobic perfusion failed to exhibit a stable LVDP >130 mm Hg or
crystalloid-perfused hearts that failed to exhibit a stable LVDP
>100 mm Hg were not entered into the study.
Expression of Results and Metabolic and
Statistical Analyses
Individual postischemic recoveries of LVDP were
expressed as percentages of their preischemic values. The
record of ischemic contracture from the isovolumic
intraventricular balloon (Fig 2)
provided a number of measures of the severity of contracture: the time
to onset of contracture, the time to peak contracture, the time to 75%
of peak contracture, and the magnitude of peak contracture (millimeters
of mercury). All hearts were stored in liquid nitrogen and lyophilized
at the end of the study. The metabolite analysis was performed
by an independent laboratory17 without knowledge of the
identity of the samples. Metabolite concentrations were expressed in
micromoles per gram of dry weight.
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All data are expressed as mean±SE. For each study involving more than two groups, a one-way ANOVA was performed, and when a significant F value was obtained, comparisons between groups were carried out with Tukey's t test. For study 2, Student's t test was used. Statistical significance was defined at P<.05. Hearts were randomized within each study.
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Results |
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LVDP at the end of the 20-minute preischemic period (see the Table
) did not differ significantly between the
groups. Less than 5% of hearts were excluded from the studies. Lactate
results were not available for three of the six preconditioned hearts
in the second group of study 3C (Fig 1
).
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Study 1: Effect of Preconditioning and/or Cardioplegia on
Ischemic Contracture and Postischemic
Contractile Recovery
Ischemic Contracture
Compared with unmodified ischemia, preconditioning greatly
accelerated whereas cardioplegia greatly delayed ischemic
contracture (Fig 3). Thus, time to 75% contracture was
reduced by preconditioning from its control value of 14.3±0.4 to
6.2±0.3 minutes (P<.05) but was increased by cardioplegia
to 23.9±0.8 minutes (P<.05). The combination of
preconditioning and cardioplegia resulted in an intermediate profile
with a time to 75% contracture of 15.4±2.4 minutes (P=NS
versus control).
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P<.05 vs PC+CP.
The magnitude of peak contracture was significantly increased by
preconditioning but was unaffected by cardioplegia (Fig 3): control,
77±3 mm Hg (P<.05 versus preconditioning);
preconditioning, 97±5 mm Hg (P<.05 versus control and
cardioplegia); cardioplegia, 75±2 mm Hg (P<.05 versus
preconditioning); and preconditioning plus cardioplegia, 86±4 mm Hg
(P=NS). However, by the end of the 35-minute period of
ischemia, contracture in all groups converged to a similar
value (control, 57±5 mm Hg; preconditioning, 66±3 mm Hg;
cardioplegia, 61±2 mm Hg; and preconditioning plus cardioplegia, 68±3
mm Hg).
Postischemic Contractile Recovery
Ischemic preconditioning, cardioplegia, and their
combination resulted in a high and generally similar protection against
contractile injury (P<.05 versus control). Thus, the
postischemic recovery of LVDP (Fig 4)
increased from 26±6% in control hearts to 44±2% in hearts with
preconditioning, 53±3% in hearts with cardioplegia, and 54±4% in
hearts with preconditioning plus cardioplegia. No clear relationship
existed between postischemic contractile recovery and any
of the indexes of contracture.
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Study 2: Effect of the Extent of Development of Ischemic
Contracture on Postischemic Contractile
Recovery
Although our results show that there is a difference among the
preconditioning, cardioplegia, and control groups in the profiles for
contracture during ischemia, identical contracture was
present in all groups by the end of ischemia. It was of
interest to determine what would have happened to functional recovery
had we reperfused earlier when the degree of contracture was very
different among groups. We therefore selectively repeated some of the
preceding studies in the blood-perfused heart but reperfused the
hearts after only 7.5 minutes of ischemia. This time was
selected on the basis of the results presented in Fig 3, which
show that contracture was minimal in the control group at this time but
maximal in the preconditioned group.
Ischemic Contracture and Postischemic
Contractile Recovery
As Fig 5A shows, after 7.5 minutes of
ischemia, the degree of contracture in the preconditioned group
was high (58±8 mm Hg), but it was only 16±4 mm Hg in the control
group (P<.05). Despite this, the postischemic
recovery of LVDP was identical in both groups (control, 80±3%;
preconditioned heart, 80±1%; P=NS; Fig 5B). However, after
this short duration of ischemia (a time known to render the rat
heart very susceptible to reperfusion
arrhythmias18 ), reperfusion-induced
ventricular fibrillation was seen in seven of eight control
hearts but in only one of eight preconditioned hearts
(P<.05).
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indicates control; 
, ischemic
preconditioning. *P<.05 vs control.
High-Energy Phosphates and Lactate
The mean ATP, phosphocreatine, and lactate contents before 7.5
minutes of ischemia were similar in preconditioned and control
hearts (14.5±1.1 versus 13.5±0.9, 13.2±2.1 versus 11.9±1.9, and
6.8±0.7 versus 6.7±0.8 µmol/g dry wt, respectively). However, at
the end of 7.5 minutes of ischemia, the ATP and lactate
contents in preconditioned hearts were significantly less than those in
control hearts (3.9±0.5 versus 7.1±0.8 and 104±2 versus 127±4
µmol/g dry wt, respectively; P<.05).
Study 3: Relationship Between the Number of Cycles of
Preconditioning and the Occurrence of Ischemic
Contracture
Blood-Perfused Hearts
Compared with unmodified ischemia, preconditioning with
just one cycle resulted in such an intensification of ischemic
contracture that increasing the number of preconditioning cycles
produced only a modest additional increase (time to 75% contracture:
14.5±0.4 minutes for control hearts and 6.0±0.2, 5.6±0.2, and
4.8±0.2 minutes for hearts subjected to one, two, and three cycles of
preconditioning, respectively; P<.05 for hearts subjected
to three versus one and two cycles; Fig 6A). Although in
blood-perfused hearts a modest frequency dependency could be
demonstrated between the number of preconditioning cycles and time to
75% and peak contracture, there was no apparent frequency dependency
for the time to onset or degree of contracture at the end of
ischemia.
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PC (2), and 
PC (3), ischemic preconditioning with
1, 2, and 3 cycles of 3 minutes of ischemia plus 3 minutes of
reperfusion, respectively. *P<.05 vs all;
Crystalloid-Perfused Hearts
The ability of preconditioning to accelerate ischemic
contracture was clearly dependent on the number of preconditioning
cycles (Fig 6B). Thus, the time to 75% contracture was as follows:
14.7±0.2 minutes for control hearts and 15.1±1.0, 11.4±1.0, and
10.6±1.4 minutes for hearts subjected to one, two, and three cycles of
preconditioning, respectively (P<.05 for all except hearts
subjected to one cycle versus control hearts; P<.05 for
hearts subjected to two and three versus one cycle). In contrast to the
blood-perfused hearts, one cycle of preconditioning had relatively
little effect on ischemic contracture, although for all groups,
increasing the number of preconditioning cycles had no effect on the
time to onset or the degree of contracture at the end of
ischemia.
When the second cycle of preconditioning ischemia was increased
to 5 minutes (Fig 6C), there was a further and substantial
intensification of ischemic contracture (time to 75%
contracture, 7.0±0.8 minutes). However, this was not increased further
by the addition of a third cycle (time to 75% contracture, 6.2±0.6
minutes). Again, increasing the duration or the number of
preconditioning cycles had no effect on the time to onset or final
degree of contracture.
The lactate content before the onset of ischemia was between 3
and 5 µmol/g dry wt. By the end of 20 minutes of ischemia,
the lactate content was reduced in proportion to the preceding
"dose" of preconditioning (and thus in parallel to the timing of
the ischemic contracture; Fig 6B and 6C). The values were as
follows: for study 3B (see Fig 1): 161±5 µmol/g dry wt for control
hearts; 151±8, 123±10, and 104±8 µmol/g dry wt for hearts
preconditioned with 1, 2, and 3 cycles; and for study 3C, 86±8
µmol/g dry wt for hearts preconditioned with 2 cycles
(P<.05, control versus all except hearts preconditioned
with 1 cycle).
Study 4: Relationship Between the Number of Cycles of
Preconditioning and Postischemic Contractile
Recovery
In the crystalloid-perfused heart, preconditioning with one
and three cycles resulted in similar protection of
postischemic contractile recovery (Fig 7).
Thus, the recovery of LVDP was increased from 37±8% in control hearts
to 64±4% with one cycle of preconditioning and 66±4% with three
cycles of preconditioning (P<.05). Similarly, the
diastolic compliance at the end of reperfusion was equally
better protected by one and three cycles of preconditioning; the
intraventricular balloon volume required to achieve
an LVEDP of 4 mm Hg (preischemic 
220 µL) was reduced
to 18±3 µL in control hearts but only to 39±9 µL with one cycle
of preconditioning and to 38±5 µL with three cycles of
preconditioning (P<.05).
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Discussion |
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Preconditioning, Cardioplegia, and Ischemic Contracture
Using the isolated blood-perfused rat heart, we have shown that compared with unprotected ischemia, preconditioning greatly accelerates ischemic contracture. This contrasts with cardioplegia, which greatly delays contracture. Combining cardioplegia with preconditioning resulted in an intermediate situation in which the profile for contracture was not dissimilar to that for control hearts. Despite this diversity in contracture profiles, all three interventions afforded a similar degree of protection of postischemic contractile function. Thus, our results show that the severity of ischemic contracture, either during or at the end of an ischemic episode, is an unreliable indicator of the ability of the heart to recover during a 40-minute reperfusion period. Because our studies were with isolated hearts (which can be studied only for relatively short lengths of time), we cannot comment on the relationship between contracture and long-term functional recovery.
Could an Accelerated Ischemic Contracture Limit the
Potential Benefit of Preconditioning?
Although preconditioning is highly protective, some of its benefit
might be lost as a consequence of possibly detrimental side effects
such as intensification of ischemic contracture. If this were
so, the benefits of preconditioning might be further enhanced if the
intensification of ischemic contracture could be
circumvented.
When the acceleration of ischemic contracture by
preconditioning was prevented by the combination of preconditioning and
cardioplegia, there was no further improvement in
postischemic contractile recovery. In study 4 (Figs 6B and 7), three preconditioning cycles increased the severity of contracture
without reducing the protection of postischemic contractile
function or diastolic compliance compared with one cycle of
preconditioning. Thus, at least in the context of preconditioning,
acceleration of ischemic contracture is not necessarily
detrimental and is certainly a poor predictor of
postischemic recovery.
Contracture as an Index of Ischemic Injury
Studies in the neonate have led a few investigators to question
the necessity for an association between ischemic contracture
and postischemic function. Thus, Riva and
Hearse11 showed that the severity of contracture was an
unreliable indicator of tissue injury in rats of various ages. Further
evidence10 19 suggests that the neonatal heart, despite
developing ischemic contracture earlier, is much more
resistant to ischemic injury than the adult heart.
However, dissenting studies19 suggest the opposite. It is
perhaps significant that the dissenting workers used measures of
diastolic state or ischemic contracture as the only
means of assessing injury and that some have, in more recent studies in
which they used ischemic contracture and contractile recovery
to assess injury, come into conflict with their earlier
conclusions.8 20
Ventura-Clapier and Veksler,21 using metabolically inhibited rat cardiac muscle fibers, have shown that the contracture induced by reducing ATP content of the superfusate was fully reversible on reintroduction of ATP. Previous evidence that ischemic contracture might not be synonymous with irreversible injury came from Steenbergen et al,22 who showed that reperfusion shortly after peak contracture resulted in full restoration of calcium and metabolic status with no significant enzyme or calcium leakage. In a series of studies, Wexler et al9 showed that intermittent mechanical stretching of a heart as it went into contracture could protect against the phenomenon. Although it would be unwise to dismiss contracture as an indicator of ischemic injury in all conditions, its validity should be critically appraised when used for this purpose.
Number of Cycles of Preconditioning in Relation to Ischemic
Contracture and Other Indexes of Tissue Injury
A relationship clearly existed between the number of
preconditioning cycles and the severity of ischemic
contracture. This may help reconcile the conflicting evidence in the
literature about the effect of ischemic preconditioning on
contracture. Thus, Banerjee et al23 found that
preconditioning with one cycle of 2 minutes of ischemia plus 10
minutes of reperfusion did not accelerate ischemic contracture
in the crystalloid-perfused rat heart, whereas others using
multiple cycles found that it did.14 15 Steenbergen et
al,24 using four cycles of preconditioning, observed no
acceleration of ischemic contracture; however, hearts were
pretreated with
1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic
acid, which is known to buffer calcium.
The frequency dependency of ischemic contracture was not paralleled by a similar dependency of postischemic functional recovery, nor was it evident on the basis of time to onset of contracture or degree of contracture at the end of the ischemic period. It is becoming increasingly clear from the literature on preconditioning that there are striking differences in the degree of dose dependency associated with different end points of injury. Thus, for reducing infarct size, no dose dependency is seen above a certain minimal dose.25 26
By contrast, both ischemia- and reperfusion-induced arrhythmia is highly dose dependent on both the duration of the preconditioning ischemia and the number of cycles.18 27 These observations raise the interesting possibility that several different mechanisms are responsible for the ability of preconditioning to influence various facets of ischemia- and reperfusion-induced injury.
These results may be unique to the rat; in other species, preconditioning might not accelerate ischemic contracture. Relevant to this is the observation that preconditioning has been shown to attenuate intracellular acidosis during ischemia in all species studied. Because a close correlation seems to exist between the development of acidosis and ischemic contracture, it would appear that our results probably are not unique to the rat.
Preconditioning and Acceleration of Contracture: Possible
Mechanisms
Although the literature is in conflict over whether an increase in
calcium precedes or follows the decline in ATP associated with
ischemic contracture,21 there is strong evidence
that the development of ischemic contracture can be linked to
tissue ATP content. Thus, it has been shown that the onset and
development of contracture can be linked very closely to the extent and
rate at which ATP declines during ischemia.3 28
This conclusion is also supported by Kupriyanov et al,29
who produced similar findings with nuclear magnetic resonance studies.
Our ATP results from study 2 in the blood-perfused rat heart also
support this conclusion.
Evidence that the onset of ischemic contracture is very closely
related to the rate of ATP production has been provided by Owen
et al,30 who used the rat heart with low-flow
ischemia. They found that as long as ATP production
during low-flow ischemia was maintained >3 µmol/g fresh
wt per minute (by a combination of anaerobic glycolysis and
residual oxidative phosphorylation), ischemic
contracture would not occur. They calculated that during
ischemia, anaerobic glycolysis could produce a
maximum of only 2.5 µmol ATP/g fresh wt per minute. Thus, during
global ischemia, when oxidative phosphorylation
ceases and ATP production from anaerobic glycolysis
is insufficient to maintain tissue energy demands, the availability of
ATP will gradually decrease, and at some concentration3 or
reduced rate of production,30 contracture will
develop. In this way, glycolytic flux may determine the development of
ischemic contracture. Thus, if anaerobic glycolysis
were halted by a lack of substrate or the presence of
inhibitors, there would be a precipitous shortfall in
available ATP, which would lead to the immediate development of
contracture.3 30 31 Kingsley et al32 offered
further collaborative evidence by demonstrating that ischemic
contracture developed when the production of protons
during ischemia (mainly from anaerobic glycolysis)
stopped (a situation that would be indicated by intracellular pH
reaching a plateau). Our lactate measurements in the blood- and
crystalloid-perfused rat heart also support this hypothesis because
we found that the acceleration of ischemic contracture was
paralleled by a decreasing lactate content.
It could be argued that the point at which the availability of ATP becomes limiting and contracture is initiated will depend on the initial tissue content of ATP and the duration of anaerobic glycolytic ATP production. Regarding the former, in preconditioned hearts, tissue ATP content at the onset of the extended period of ischemia is likely to be similar after a number of preconditioning cycles and not too dissimilar from that in control hearts when the period of preconditioning ischemia is as brief as that used in these studies.14 33 The latter would be influenced by various factors, including residual flow, endogenous glycogen supplies, and the availability of exogenous substrate. Because anaerobic glycolysis is stimulated after as little as 10 seconds of ischemia and principally involves consumption of glycogen,34 it is likely to be stimulated substantially and rapidly during each ischemic episode of preconditioning, and endogenous glycogen is likely to be progressively reduced.35 Thus, in the preconditioned heart, at the onset of the extended period of ischemia, glycolytic flux is likely to be similar compared with control hearts, but the period for which it may be sustained may be reduced owing to a reduction of endogenous glycogen. Consequently, one might predict an initial rate of intracellular acidosis similar to that in control hearts, a reduction in the final degree of acidosis, and (if contracture is indeed triggered by a cessation of glycolytic ATP production) an earlier contracture. Support for this concept comes from a number of studies in which intracellular or extracellular acidosis was used to assess glycolytic activity.13 14 29 32 These show that the initial rate of decline of pH during ischemia in control, glycogen-depleted, metabolically inhibited, and preconditioned hearts is initially similar. In all instances, pH falls to a plateau that in the preconditioned, glycogen-depleted, and metabolically inhibited hearts is at a higher level than that in the control hearts. Further evidence comes from studies of the rate of lactate production, which again shows an initial similarity but with a plateau that is reached sooner in preconditioned hearts.33 It has also been shown that metabolic inhibition and glycogen depletion accelerate ischemic contracture.13 14 29 32 36 Hence, a linear relationship seems to exist among the capacity for anaerobic glycolysis, timing of ischemic contracture, onset of the pH plateau, and final intracellular pH during ischemia.29 32
Thus, as suggested above, preischemic depletion of glycogen stores by preconditioning or other interventions will result in anaerobic glycolysis being maintained for a shorter time, an acceleration in the development of ischemic contracture, and reduced development of intracellular acidosis.29 32 Volovsek et al35 showed that the degree of glycogen depletion increases with increasing numbers of preconditioning cycles. Taken together, one would expect to see frequency and time dependencies in the ability of preconditioning ischemia to influence contracture. These were seen in the present studies. The similarities in the frequency dependency of preconditioning in relation to both arrhythmia and contracture might suggest a similarity in the underlying mechanism. Associated changes in calcium distribution and the limitation of acidosis would provide suitable conditions for explaining the potent antiarrhythmic consequences of preconditioning.
When cardioplegia was given to preconditioned hearts, acceleration of ischemic contracture was not observed, nor was it delayed as in hearts given cardioplegia alone. Using nuclear magnetic resonance, we have shown37 that cardioplegia delayed the depletion of high-energy phosphates and the onset of intracellular acidosis. If, as Kingsley et al32 and Eisner et al38 suggest, the onset of intracellular acidosis correlates with the onset of anaerobic glycolysis, then cardioplegia also would be expected to delay the latter. Thus, cardioplegia given to preconditioned hearts might be expected to delay the onset of anaerobic glycolysis so that even though there is reduced amount of substrate, it will come to a stop later than in preconditioned hearts without cardioplegia. Consequently, one might expect the need for provision of anaerobic ATP to be postponed, as well as the development of ischemic contracture. We observed the latter.
The mechanism proposed to explain the ability of preconditioning to accelerate contracture has been presented on the basis of the supposition that either directly or indirectly, ATP availability is a critical determinant of the phenomenon. Although other preconditioning-induced changes such as those in intracellular pH and inorganic phosphate might influence the magnitude of peak contracture (through modifications of myofibrillar calcium sensitivity), they are unlikely to influence the time course of the contracture.21
Conclusions
Our studies have shown that despite intensifying ischemic
contracture, preconditioning can protect postischemic
contractile recovery. The intensification of ischemic
contracture does not appear to be an unfavorable side effect of
preconditioning that subtracts from its clear benefit. Finally, our
results lead us to question the validity of contracture as an index of
ischemic injury.
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Acknowledgments |
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This work was funded in part by grants from STRUTH and the British Heart Foundation. The advice and discussion of Drs M.J. Shattock, M. Avkiran, and D. Chambers are gratefully acknowledged.
Received May 1, 1995; revision received November 6, 1995; accepted November 19, 1995.
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