Dichotomy of Ischemic Preconditioning
Improved Postischemic Contractile Function Despite Intensification of Ischemic Contracture
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.
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.
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
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).
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.
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.
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.
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.
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.
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.
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⇑).
Study 1: Effect of Preconditioning and/or Cardioplegia on Ischemic Contracture and Postischemic Contractile Recovery
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).
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.
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).
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
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.
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).
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
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.
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.
- Copyright © 1996 by American Heart Association
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