Ischemic Preconditioning Does Not Protect Against Contractile Dysfunction in the Presence of Residual Flow
Studies in the Isolated, Blood-Perfused Rat Heart
Background We have previously demonstrated that ischemic preconditioning (PC) does not protect when oxygen deprivation is accompanied by a high level of perfusion (hypoxia). Since clinical ischemia can vary from mild to severe, we wished to determine whether PC could protect against injury arising from low-flow ischemia.
Methods and Results Functional recovery after 30 minutes of reperfusion was assessed in isolated, blood-perfused rat hearts (n=6 per group) subjected to (A) 30 minutes of zero-flow ischemia, (B) 30 minutes of zero-flow ischemia preceded by 3×PC (PC=5 minutes of ischemia+5 minutes of reperfusion), (C) 90 minutes of low-flow ischemia at 10% of baseline coronary flow (0.31±0.02 mL/min per gram wet wt), (D) 90 minutes of low-flow ischemia at 10% of baseline coronary flow (0.29±0.02 mL/min per gram wet wt) preceded by 3×PC. PC significantly protected against injury resulting from zero-flow ischemia (developed pressure recovered to 67±6% versus 31±12% in B and A, respectively; P<.05) but not resulting from low-flow ischemia (recovery of developed pressure was 40±8% versus 37±7% in C and D, respectively). Protein kinase C (PKC) is widely considered to be involved in the mechanism of PC such that prior activation and translocation of PKC by the PC protocol allows phosphorylation of the end-effector protein early during the subsequent ischemic insult, before loss of adenosine triphosphate occurs. However, because adenosine triphosphate content falls slowly during low-flow ischemia, PKC may be activated and translocated early enough to be active during this insult. If so, inhibition of PKC should decrease functional recovery in the control group. However, functional recovery in control groups was not decreased in the presence of the PKC inhibitor polymyxin B (50±6%), suggesting that if activation of PKC occurred during low-flow ischemia, it was not protective.
Conclusions PC does not protect against contractile dysfunction in the rat when a low level (10% of baseline flow) of ischemic perfusion remains during the prolonged insult.
Previously we and others have demonstrated that ischemic preconditioning significantly improves functional recovery after prolonged zero-flow ischemia in the isolated rat heart.1 2 3 However, preconditioning did not protect against contractile dysfunction when oxygen deprivation was accompanied by a high level of perfusion (hypoxia).4
The mechanism by which preconditioning provides protection against ischemia-induced injury is unclear. Initiation of protection appears to be receptor mediated,5 6 specifically by receptors linked to the second messenger protein kinase C (PKC),3 7 8 but how PKC mediates the protection is still unclear. However, it does appear that receptor occupation is important during both the preconditioning ischemia and the prolonged ischemic insult.9 The A1-specific antagonist PD 115199 completely blocked preconditioning-induced protection when it was infused either before or after the preconditioning ischemia.9 Thus the lack of protection against hypoxia-induced injury4 may be because preconditioning-induced protection requires the accumulation of a factor in the ischemic myocardium, either to exert the preconditioning protective effect (eg, adenosine) or as a factor of injury against which preconditioning affords protection (eg, protons).
Clinical ischemia can vary in severity, even in the setting of myocardial infarction. For example, in most patients, after an acute coronary occlusion, myocardial perfusion in the infarct region is ≤15% of normal coronary flow.10 We wished to determine therefore whether preconditioning could protect against a prolonged period of low-flow ischemia at such a level of ischemic perfusion.10 To better simulate the perfusion conditions and metabolic milieu in the region of an acute myocardial infarction, we used the isolated, blood-perfused rat heart model with low-flow ischemia where, in contrast to hypoxia, full washout of metabolites (such as protons) produced during low-flow ischemia does not occur. In previous studies we have demonstrated that intracellular pH falls to ≈6.1 during a sustained period of low-flow ischemia imposed at 10% of baseline coronary flow11 ; in addition, although partial lactate washout occurs during the ischemic period, there is a large peak immediately upon reperfusion when the lactate, which has accumulated during the low-flow ischemia, is washed out.11
Thus the initial aim of this study was to determine if ischemic preconditioning could protect against contractile dysfunction arising from a prolonged period of low-flow ischemia. Our results (see below) demonstrated that the presence of a residual level of ischemic perfusion eliminated the protection conferred by preconditioning against zero-flow ischemia. To further investigate the mechanism by which low-flow ischemia eliminated preconditioning-induced protection, we determined whether inclusion of the protective ischemic metabolite adenosine12 could restore preconditioning-induced protection and whether PKC may be activated and protective during low-flow ischemia in control hearts, thereby negating the difference between control and preconditioned hearts.
Hearts were obtained from male Sprague-Dawley rats (weight, 250 to 300 g). The isolated rat heart preparation, perfused with modified Krebs-Henseleit buffer containing bovine red blood cells at a hematocrit of 40%, was used; this methodology has been described previously.13 Rats were anesthetized with sodium pentobarbital (0.1 mL/100 g IP injection), heparinized (sodium heparin, 100 IU/100 g IV), and the heart rapidly excised. The aorta was then cannulated and coronary perfusion initiated and adjusted during the initial oxygenated perfusion to achieve a steady coronary perfusion pressure of 80 mm Hg. After equilibration, flow was then held constant and coronary perfusion pressure was determined by coronary vasomotor tone. The left ventricle was vented of thebesian drainage through an apical cannula and the pulmonary artery cannulated to allow collection of coronary sinus flow for measurement of coronary flow rate and for assessment of myocardial oxygen consumption (MVo2) and lactate production. A fluid-filled balloon connected to a Statham P23db pressure transducer (Gould) was inserted into the left ventricle to measure left ventricular developed pressure (LVDP). The balloon was filled until a left ventricular end-diastolic pressure (LVEDP) of 10 mm Hg was achieved and the balloon volume then held constant such that changes in LVEDP reflected changes in left ventricular diastolic distensibility.14 All hearts were paced throughout the experiment at 6 Hz except for the periods of zero-flow ischemia and the first 3 minutes of reperfusion thereafter. The heart was placed in a water-jacketed constant temperature chamber maintained at 37°C and submerged in saline. Hearts were excluded from study if baseline LVDP was <100 mm Hg. Low-flow ischemia was imposed by first reducing coronary flow to 20% of baseline flow for 5 minutes and then to 10% of baseline flow; this level of ischemic perfusion was then maintained for the remainder of the ischemic period. Hearts were reperfused at the baseline coronary flow rate.
All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institute of Health (NIH Publication No. 86-23, revised 1985).
A red blood cell perfusate similar to that described previously13 was used, consisting of bovine red blood cells at a final hematocrit of 40% in Krebs-Henseleit buffer. Fresh cow blood was collected into a vessel containing ≈6000 units of sodium heparin and 100 000 units of penicillin per liter. The blood was centrifuged at 5°C at 3000 rpm for 15 minutes. The supernatant was aspirated and the resulting packed cells were mixed 1:1 with Krebs-Henseleit buffer. The centrifugation and resuspension steps were repeated three times. The red blood cell suspension was thus essentially free of white blood cells and platelets. Packed red blood cells were stored in nominally calcium-free buffer at 4°C and washed daily before use. Cells more than 4 days old were not used.
The modified Krebs-Henseleit buffer contained (in mmol/L): NaCl 118, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25.5, glucose 5.5, lactate 1.0, and palmitic acid (as a source of free fatty acid) 0.4, in combination with 4 g% bovine serum albumin (No. A7030, Sigma Chemical Co). Essentially fatty acid–free bovine serum albumin was first dissolved in Krebs-Henseleit buffer. Palmitic acid (No. P-9767, Sigma Chemicals Co) was then added to this mixture. Calcium chloride was gradually added to the final perfusate mixture to achieve a calcium-free concentration of ≈1.2 mmol/L. Gentamicin (2 mg/L) was added to the red blood cell perfusate to retard bacterial growth. The perfusate was equilibrated with 20% O2/3% CO2/77% N2 to achieve a Po2 of 140 mm Hg and a pH of 7.4.
Adenosine was dissolved in saline and infused into the aortic cannula, immediately above the heart, at 5% of the coronary flow rate to achieve a final concentration of 100 μmol/L in the blood. Similarly, the PKC inhibitor polymyxin B was dissolved in the Krebs-Henseleit buffer and also infused into the aortic cannula, immediately above the heart, at 5% of the coronary flow rate to achieve a final concentration of 50 μmol/L in the blood. We have previously demonstrated that this concentration of polymyxin B successfully abolished preconditioning-induced protection against zero-flow ischemia.7 Polymyxin B had no effect on the Pco2, Po2, or pH of the blood.
Data Collection and Analytical Procedures
Coronary blood flow was measured by timed collections of the coronary venous effluent through the pulmonary artery. MVo2 was calculated from the arteriovenous oxygen content differences, derived from oxygen saturation curves for the Krebs buffer/red blood cell suspensions over the experimental range of pH and Po2 values. Arterial and venous lactate concentrations were measured with the use of the COBAS BIO system (Roche Diagnostic Systems). Before the assay, the lactate samples were diluted 1:3 with 10% trichloroacetic acid and stored at 4°C.
All hearts were subjected to 30 minutes of normoxic perfusion before either (a) a further 30 minutes of normoxic perfusion (control groups) or (b) 3×(5 minutes of ischemia and 5 minutes of reperfusion) (preconditioned groups). Hearts were then subjected to either 30 minutes of zero-flow global ischemia and 30 minutes of reperfusion or 90 minutes of low-flow ischemia (10% of baseline flow) and 30 minutes of reperfusion. Contractile function, myocardial oxygen consumption, creatine kinase release, and lactate release were assessed throughout.
Adenosine or polymyxin B was infused for 5 minutes before the onset of low-flow ischemia and thereafter for the remainder of the experiment.
The control and preconditioned groups subjected to zero-flow ischemia were concurrently perfused with the low-flow groups but have been previously published7 and are reported here as a comparison for the groups subjected to low-flow ischemia.
Data are reported as mean±SEM. Data acquired sequentially in individual hearts were tested by a two-way ANOVA for repeated measures. If this analysis indicated a significant difference between groups, values at specific time points were examined by a method of least significance. A value of P<.05 was considered significant. Differences in mean measurements between experimental groups was tested by a two-tailed unpaired t test or one-way ANOVA followed by the Bonferroni test.
Determination of Whether Preconditioning Could Protect Against Injury Induced by a Prolonged Period of Low-Flow Ischemia and Reperfusion
Four groups of hearts (n=6 per group) were studied: control and preconditioned hearts after 30 minutes of zero-flow ischemia and 30 minutes of reperfusion and control and preconditioned subjected to 90 minutes of low-flow ischemia and 30 minutes of reperfusion. The period of low-flow ischemia was chosen such that the percentage recovery of LVDP in the two control groups (zero- and low-flow ischemia) would be similar, that is, both control groups would suffer a similar degree of ischemic damage. No significant differences were observed between the groups in terms of preischemic absolute values of contractile function, coronary flow, or coronary perfusion pressure (Table 1⇓).
In both preconditioned groups, the preconditioning protocols had similar effects. Developed pressure fell to ≈60 to 70 mm Hg (Fig 1⇓; zero-flow data shown for comparison) and end-diastolic pressure fell to 4.8±0.8 and 5.4±1.3 mm Hg in the zero-flow and low-flow preconditioned groups, respectively, after the preconditioning protocol (Fig 2⇓; zero-flow data shown for comparison).
After the prolonged ischemic insult, preconditioning was protective only after zero-flow ischemia (Fig 1⇑, left). Recovery of LVDP was 67±6% and 31±12% in preconditioned and control groups, respectively. No preconditioning-induced protection was observed after low-flow ischemia (Fig 1⇑, right) in which recovery of LVDP was 37±7% and 40±8% in preconditioned and control groups, respectively. Similarly, a marked protection against diastolic dysfunction was observed in the preconditioned group after zero-flow ischemia (Fig 2⇑, left); this protection was completely lost after low-flow ischemia (Fig 2⇑, right).
Lactate release after the prolonged period of zero-flow ischemia was significantly decreased in the preconditioned group compared with its control (Fig 3⇓, left). In contrast, no reduction in total lactate release was seen in preconditioned hearts after low-flow ischemia and reperfusion (Fig 3⇓, right).
Can Infusion of Adenosine, an Endogenous Initiator of Preconditioning, Restore Preconditioning-Induced Protection Against Injury Sustained During Low-Flow Ischemia?
Liu and colleagues6 have demonstrated that adenosine is required not only to initiate preconditioning-induced protection in the rabbit but also to mediate the protection. If an adenosine antagonist is infused after the preconditioning cycles of ischemia and reperfusion but immediately before the sustained ischemic period, protection is lost. This demonstrates that adenosine receptors must be occupied during both the preconditioning protocol and the prolonged ischemic insult for preconditioning to be effective. Furthermore, Headrick15 recently reported that pretreatment with cyclohexyladenosine mimicked preconditioning and that the competitive adenosine receptor antagonist 8-(p-sulfophenyl) theophylline abolished preconditioning in the isolated rat heart. Although the role of adenosine in preconditioning in the rat heart is controversial,16 17 18 it is possible that the failure to observe preconditioning-induced protection after low-flow ischemia was because the necessary mediator, adenosine, was washed out.
No significant differences were observed between the control and preconditioned groups in terms of preischemic absolute values of contractile function, coronary flow, or coronary perfusion pressure (Table 2⇓). No prolonged changes were observed on the start of adenosine infusion, although a transient dip of about 10 mm Hg in coronary perfusion pressure was observed. No heart rate effects were observed (pacing was continued throughout the protocol).
As in the previous groups, the preconditioning protocol resulted in a fall in developed pressure to 73±4 mm Hg (Fig 4⇓) and end-diastolic pressure to 5.0±0.6 mm Hg after the third episode of ischemia and reperfusion.
As in the previous groups, no preconditioning-induced protection was observed either during or after the prolonged low-flow ischemic insult (Fig 4⇑). Recovery of LVDP was 37±7% and 40±8% in preconditioned and control groups, respectively. Thus the presence of adenosine did not restore the protective effect of preconditioning during low-flow ischemia.
Does the PKC Inhibitor Polymyxin B Decrease Contractile Recovery After Low-Flow Ischemia and Reperfusion in Control Hearts?
An alternative explanantion for the absence of preconditioning-induced protection against injury sustained during low-flow ischemia was that PKC was activated during the initial few minutes of low-flow ischemia. In contrast to zero-flow ischemia, phosphorylation potential may remain high enough, for long enough, in low-flow ischemia for PKC to phosphorylate the appropriate end-effector protein. If this were the case, it is possible that under conditions of low-flow ischemia, all hearts, that is, control and preconditioned, are effectively “preconditioned.” To test this theory, we repeated the control group protocol (90 minutes of low-flow ischemia and 30 minutes of reperfusion) in the presence of the PKC inhibitor polymyxin B (50 μmol/L). If PKC was activated and protective against injury sustained during low-flow ischemia, functional recovery should be decreased in the presence of polymyxin B compared with control hearts perfused in the absence of polymyxin B. In initial studies when polymyxin B was dissolved in the buffer, coronary flow rate was significantly reduced. This had particular consequences for the low-flow insult because the low-flow ischemia was imposed at 10% of baseline coronary flow rate. In addition, preischemic function was significantly decreased. To avoid this, polymyxin B was infused at 5% of the coronary flow rate into the aortic cannula, immediately above the heart, starting 5 minutes before the low-flow insult, and the low-flow ischemic flow rate was therefore calculated on the basis of baseline flow in the absence of polymyxin B. No significant differences in ischemic flow rate were then observed compared with a second currently performed control group (Table 3⇓).
From Table 3⇑, it can be seen that percentage recovery of LVDP in the control group in the absence (55±12%) and presence of polymyxin B (56±1%) was similar.
In this study, we have demonstrated that in the isolated, blood-perfused rat heart, preconditioning does not protect against contractile dysfunction when residual flow is present during the sustained insult. This failure to demonstrate protection was not due to a washout of the ischemic metabolite adenosine nor due to a protective effect of PKC, which may have been activated early enough during low-flow ischemia (in the control group) to phosphorylate the end-effector protein. In a previous study, we have also demonstrated that preconditioning was unable to protect against contractile dysfunction resulting from a prolonged period of hypoxia,4 that is, continued coronary flow with hypoxic buffer.
The reason for this loss of protection against hypoxic or low-flow ischemia–induced injury is unknown but may be related to effects of tissue perfusion or washout. In the previous study, where the sustained insult was hypoxia, flow was maintained at normoxic levels and thus complete washout of all ischemic metabolites probably occurred.4 In contrast, in the current study, washout of ischemic metabolites was undoubtedly incomplete. In the isolated, blood-perfused rat heart model, flow rates are at a physiological level of between 2 and 3 mL/min per gram wet wt. Consequently, when coronary flow rate is reduced to 10% of baseline level, the residual flow is only 0.2 to 0.3 mL/min per gram wet wt. In a previous study, we have demonstrated that under these conditions, washout of protons is incomplete.11 Intracellular pH decreased to 6.1, a similar level to that observed during zero-flow ischemia.2 Similarly, only a partial washout of lactate occurs; upon reperfusion, a large lactate washout peak was observed during the first minute of reperfusion, indicating that a significant portion of the lactate produced during ischemia remains in the tissue until reperfusion. Thus it is likely that a significant proportion of other ischemic metabolites, such as adenosine, also remain in the tissue. Despite this, however, preconditioning was unable to protect against injury sustained during a low-flow insult.
To investigate the influence of tissue washout further, we infused 100 μmol/L of adenosine during the sustained period of ischemia. Adenosine was chosen because it is one of the few ischemic metabolites known to initiate preconditioning but that could be infused without metabolic cost during low-flow ischemia. If, for example, an α1-agonist such as phenylephrine had been infused, contractile function would have been stimulated and at this level of perfusion the degree of relative ischemia exacerbated. Furthermore, Headrick15 recently reported that the adenosine A1-receptor agonist N6-cyclohexyladenosine mimicked preconditioning and the competitive adenosine receptor antagonist 8-(p-sulfophenyl) theophylline abolished preconditioning in the isolated rat heart.15 It should be noted that these results are in contrast to others in the literature.16 17 18 The infusion of adenosine had no contractile effects and only caused a transient vasodilation that had recovered before the initiation of low-flow ischemia. The infusion of adenosine, however, failed to restore preconditioning-induced protection in this model. Thus if the washout of an ischemic metabolite is responsible for the loss of preconditioning in the rat, it is unlikely to be adenosine.
Several studies have demonstrated that glycolysis is slowed in preconditioned hearts during the sustained ischemic period.4 19 However, we have recently demonstrated that a reduction in lactate release (as a measure of glycolytic rate) is not a prerequiste for preconditioning-induced protection.7 Thus the lack of a significant reduction in lactate production in preconditioned hearts subjected to low-flow ischemia is unlikely to account for the lack of protection. Furthermore, these data are complicated by the fact that during zero-flow ischemia, lactate production will arise from glycogen breakdown, whereas during low-flow ischemia it will arise from glucose in the perfusate.
Another possible cause for the loss of protection against either ischemia or hypoxia may be that PKC may be activated early enough during the sustained insult to phosphorylate the end-effector protein. Since the translocation of PKC into the cell membrane requires ≈10 minutes20 and kinase activity parallels translocation, there is an ≈10-minute delay from activation of PKC to phosphorylation of a substrate. During zero-flow ischemia, therefore, high-energy phosphate stores will have been significantly depleted, thus limiting any protective effect. During low-flow ischemia, however, we have previously demonstrated that adenosine triphosphate (ATP) and phosphocreatine (PCr) falls far more slowly than during zero-flow ischemia; after 60 minutes, ATP and PCr content was still 41±3% and 36±3% of baseline levels, respectively.11 Thus PKC, activated and translocated early during the low-flow ischemic period, could probably phosphorylate its substrate even in control hearts. Control hearts would thus effectively be “preconditioned.” In support of this argument, both hypoxia and low-flow ischemia are able to initiate the preconditioning pheneomenon in the isolated rat heart,21 and we have previously demonstrated that the PKC inhibitor polymyxin B is able to abolish preconditioning in the isolated rat heart.7 If control hearts are indeed “preconditioned” by low-flow ischemia, then functional recovery should be decreased in the presence of polymyxin B. However, this was not the case; functional recovery was almost identical in the presence and absence of polymyxin B. Thus it is unlikely that the lack of preconditioning-induced protection against low-flow ischemia is a result of an early activation of PKC during the sustained low-flow ischemic period.
The residual ischemic flow of ≈0.3 mL/min per gram wet wt in our isolated rat heart study must be interpreted in the context of prior work that has demonstrated that preconditioning decreases infarct size in many species in which collateral flow exists.19 22 23 However, in such experimental studies, animals were excluded from study when the residual collateral flow was >0.15 mL/min per gram wet wt22 23 or 0.12 mL/min gram wet wt.19 In fact, collateral flow in the subendocardial region, where the infarct develops, was considerably less than this (≈0.04 mL/min per gram wet wt).22 Thus preconditioning can protect against low-flow ischemia but only when the degree of ischemia is at the requisite level of severity. Our results would suggest that preconditioning cannot protect against injury induced by less severe degrees of ischemia. Similar observations were made by Murry et al,19 who observed no preconditioning-induced protection against necrosis in the subepicardial zone in the dog heart, in which collateral flow ranged from 0.22 to 0.37 mL/min per gram wet wt. Murry et al19 hypothesized that the failure of preconditioning to protect these areas may be either because the preconditioning protocol failed to effectively precondition these areas, that is, the higher collateral flow in these areas would result in less damage, or because preconditioning-induced protection is only effective in severely ischemic tissue. Our results would suggest that the latter explanation is the correct one. However, the failure of preconditioning to protect against mild to moderate ischemia does not negate its proven protection against necrosis arising from severe ischemia.
Preconditioning can only delay cell death. Thus if the ischemic duration is greatly extended,19 preconditioning-induced protection is overwhelmed. However, if the rate of development of ischemic damage is slowed, for example, with hypothermia24 or cardioplegic arrest,25 preconditioning can protect over ischemic durations as long as 200 minutes. Consequently, the protocol used in the current study was carefully chosen such that control hearts recovered to a similar extent in both groups. Therefore, since injury develops slower in the presence of residual flow, hearts subjected to low-flow ischemia had to be exposed to a longer (90 minutes) sustained insult than the hearts subjected to zero-flow ischemia (30 minutes). In fact, even under conditions of normothermic ischemia, preconditioning has been shown to protect against zero-flow ischemia insults of up to 90 minutes in length.26 27 28 Thus we are confident that preconditioning did not “wear off,” nor was the protection overwhelmed during the prolonged period of low-flow ischemia. Furthermore, we have not measured PKC activity in this study, nor do we have any direct evidence that polymyxin B inhibited PKC activity. However, in previous studies using the same model, we have demonstrated that polymyxin B abolished preconditioning-induced protection.7
We have demonstrated that in the presence of a residual flow of 0.29±0.02 mL/min per gram, preconditioning failed to protect against contractile dysfunction assessed upon reperfusion. The failure of preconditioning to protect against this insult was not a result of the washout of adenosine or a result of the activation of PKC. It remains to be determined whether the loss of preconditioning-induced protection in the presence of residual flow during the sustained insult reduces the therapeutic potential of the preconditioning phenomenon.
Dr Cave was supported by the Massachusetts Affiliate of the American Heart Association. The authors gratefully acknowledge the advice of and discussions with Drs Franz Eberli and Thomas Suter.
E mail firstname.lastname@example.org
- Received December 31, 1996.
- Revision received May 9, 1997.
- Accepted May 28, 1997.
- Copyright © 1997 by American Heart Association
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