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Enzyme release during myocardial anoxia: A study of metabolic protection
Journal
of Motecatur
and Cellular
Enzyme
Def>artment
Cardiology
(1975)
7,463-482
Release During Myocardial Anoxia A Study of Metabolic Protection
D. J. HEARSE AND S. M. HUMPHREY of Biochemistry, Imfierial College of Science, Kensington,
(Received
26 October
1973,
accebted in revisedform
24 June
:
London,
U.K.
1974)
D. J. HEARSE AND S. M. HUMPHREY. Enzyme Release During Myocardial Anoxia: A Study of Metabolic Protection. Journal of Mole&r and Cellular Cardiology (1975) 7, 46% 482. In studies with the isolated perfused anoxic rat heart, myocardial enzyme release was used as an index of cellular damage. Anoxic damage (with K+ arrest) was characterized by measuring the release of four enzymes (creatine phosphokinase, a-hydroxybutyrate dehydrogenase, glutamate oxaloacetate transaminase and adenylate kinase). The possibility of protecting the myocardium against anoxic damage by using potential metabolic protective agents was investigated. The inclusion of glucose as an anaerobic energy source in the perfusion fluid during the entire anoxic period afforded considerable metabolic protection, the period of anoxia that could be tolerated without significant enzyme release was extended and the overall release was reduced. In addition, glucose during anoxia was able to confer protection against the exacerbation of enzyme release induced by reoxygenation. The results with graded hypoxia were similar to those with glucose and the two were additive. An increasing availability of oxygen afforded an increasing degree of protection. The membrane stabilizing drug methyl prednisolone had a variable effect upon enzyme release but was unable to confer true protection with increased viability upon the myocardium. The exacerbation of enzyme release induced by reoxygenation was further investigated. Potential protective agents (glucose, methyl prednisolone and the anti-oxidant ascorbate) introduced at the time of reoxygenation had little protective effect. Stepwise reoxygenation studies revealed that the extent ofexacerbation was determined by the concentration of 0s used in the reoxygenation process, the higher the 0s tension the greater the exacerbation of release. It is proposed that the onset of major myocardial enzyme release reflects the transition from reversible to irreversible cellular damage and that the action of metabolic protective agents is to prevent the deterioration of the cells to a point where they become susceptible to irreversible damage. Once the cells enter a state of irreversible damage and major enzyme release occurs then metabolic protection becomes ineffective. The results underline the importance of the need for the rapid introduction of protective agents to the anoxic myocardium. In this experimental model, protection must be initiated before the transition from reversible to irreversible cellular damage and this may possibly be best achieved either by reducing cellular energy demands or by maximizing cellular energy production. KEY WORDS: Anoxia; Hypoxia; Metabolic protection; Glucose; K+ arrest; Enzyme release; Isolated perfused rat heart; Ascorbate; Methyl prednisolone; Reoxygenation; Creatine phosphokinase; Glutamate oxaloacetate transaminase; Adenylate kinase (myokinase); cr-hydroxybutyrate dehydrogenase; Reversible and irreversible damage.
1. Introduction The appearance in the serum of specific cardiac enzymes has long been used in the diagnosis and assessment of myocardial infarction [3, 251. The leakage of enzymes
464
D.
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HEARSE
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S. M.
HUMPHREY
from the cell has been associated with biochemical and morphological damage and the extent of enzyme release is related to the severity of cellular damage. From studies of myocardial enzyme depletion in experimentally infarcted dogs it has been suggested [13,23,24] that enzyme release may be used to accurately quantitate the extent of cellular damage. Therapeutic interventions designed to protect the myocardium against damage would be expected to reduce or delay enzyme release and conversely, any extension of damage should lead to an increase in enzyme release. When cellular anoxia, hypoxia or ischemia is suspected, the aim of any intervention is to prevent the onset of irreversible cellular damage. Under conditions of cellular oxygen deprivation, myocardial metabolism is disrupted and despite the activation of anaerobic pathways, cellular energy production is severely impaired. From studies with a variety of experimental models it has been shown that following the onset of anoxia or ischemia myocardial ATP levels decline [4,5, IS, 211 and the cells enter a state of negative energy balance. The availability of adequate supplies of ATP is important not only for contractile activity but also for the maintenance of cellular integrity. It has been suggested [S, 161 that the supply of ATP is a major determinant of cellular survival and recovery and that once the level of myocardial ATP drops below a certain critical level the heart becomes irreversibly damaged and recovery is no longer possible. Associated with the critical reduction of ATP and onset of irreversible damage is the release of enzymes from the myocardium [S]. Effective metabolic protection, preventing or delaying the onset of irreversible damage may possibly be achieved with interventions specifically aimed at maintaining cellular supplies of ATP (for example by providing exogenous glucose supplies for glycolytic ATP production, or by promoting partial oxidative metabolism of endogenous substrates with a limited supply of oxygen). An alternative approach would be to attempt to prevent the loss of cellular enzymes and metabolites by the use of membrane stabilizing drugs, for example methyl prednisolone. The objective of the studies described in this paper was to assess the feasibility of preventing or delaying the onset of anoxia-induced cehular damage with the use of the potential metabolic protective agents described above. In these studies, the release of several enzymes from the isolated perfused rat heart was used as an index of the onset and the severity of myocardial damage. In the light of our recent findings [a] that under certain circumstances reoxygenation of the severely anoxic myocardium can greatly exacerbate enzyme release, we have also investigated the efficacy of these and other potential protective agents (for example gradual reoxygenation or the use of an antioxidant such as ascorbate) in preventing or reducing the oxygen-induced exacerbation of enzyme release. 2. Materials Male
rats (280 to 320g body
and
Methods
wt) of the Sprague-Dawley
strain,
maintained
on a
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standard diet, were used throughout these experiments. All substrates and enzymes used in the analysis were obtained from the Boehringer Corporation (London) Ltd. Methyl prednisolone was generously donated by the Upjohn Company (U.K.) Ltd.
Perfusion techniques Rats were lightly anaesthetized with diethyl ether, the left femoral vein was exposed and heparin (200 IU) was administered intravenously. One min after the administration of heparin, the heart was excised [20] and placed in ice cold perfusion medium until contraction had ceased. The heart was then mounted on the perfusion apparatus. The mounting procedure and apparatus were based on modifications [5] of that described by Neely et al. [20]. The perfusion apparatus was designed so that it could be used for two modes of perfusion which could be readily interconverted.
Non-working
Langendor$peparation
[ 171
Hearts were perfused via the aorta at a perfusion pressure of 100 cm of water. This preparation was used for all enzyme release studies and for the anoxic period in the functional recovery studies.
Working preparation Here hearts were perfused via the left atrium as described by Neely et al. [20] at an atria1 filling pressure of 20 cm of water. The left ventricle spontaneously ejected 40 to 50ml of perfusate/min against a hydrostatic pressure of 100 cm of water. The aortic flow and coronary flow were pooled and recirculated.
Perfusion medium Krebs-Henseleit bicarbonate buffer [14] pH 7.4 was the standard perfusion fluid. In some experiments glucose (11.1 mM) or methyl prednisolone (0.01 mM) or sodium ascorbate (1 .O mM) was included in the buffer. During K+ arrest, the concentration of K+ in the buffer was increased to 16 mM. In high K+ and also sodium ascorbate buffers the concentration of Naf was correspondingly decreased. Precautions [28] were taken to prevent the precipitation of Gas+. Before use, the perfusion fluid was filtered through a cellulose acetate filter of pore size 5.0 km
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(Millipore Ltd.). The perfusion fluid was maintained at 37°C and in aerobic or reoxygenation studies the fluid was equilibrated with 02+COz (95:5, aortic 02 partial pressure was over 600 mm Hg), in anoxic studies the fluid was equilibrated with Na+COa (95 :5, aortic 02 partial pressure was less than 5 mm Hg). In hypoxic studies or gradual reoxygenation studies the fluid was equilibrated with N2+02+ CO2, CO2 was maintained at a constant level of 5% while the relative proportions of 02 and N2 were varied to suit the experimental requirement. Atmospheric gas contamination of the heart was prevented by completely enclosing the heart in a waterjacketed (37°C) c h am b er which was continually gassed (20 ml/min) with the same gas mixture as that used for the perfusion fluid.
02 partial pressure measurement This was measured using 02 electrodes (type E5046; Radiometer, Copenhagen, Denmark). 02 partial pressure was continuously monitored using a digital acidbase analyser (type PHM72 ; Radiometer).
Tim8 sequenceof perfwion Immediately after mounting, the heart was perfused (standard perfusion fluid plus glucose at a perfusion pressure of 100 cm of water) aerobically as a non-working Langendorff preparation for a 5 min wash-out and equilibration period. For enzyme release studies, the hearts were maintained as non-working Langendorff preparations for the duration of the experiment. By utilizing a second pregassed perfusion fluid reservoir equipped with a constant head device, the hearts were then subjected to experimental perfusion periods of up to 7 h. In reoxygenation studies additional reservoirs were available for the introduction of suitably gassed perfusion fluid. During the entire experimental time course the coronary flow was recorded and samples of the coronary effluent were taken at 5 min intervals. Immediately after sampling the aliquots were assayed or were stored at 4°C for the duration of the perfusion period. During the enzyme release studies it was decided to potassium arrest the hearts for the duration of the experimental period. There were two reasons for this decision. Firstly, anoxia and hypoxia alone would rapidly induce cardiac arrest, however, arrest would be incomplete and irregular bursts of contractile activity would occur. In addition to making it difficult to standardize perfusion conditions, these anoxic bursts of activity have been associated with some enzyme release (D. J. Hearse and S. M. Humphrey, unpublished results). Secondly, in reoxygenation studies in the absence of K+ arrest, reoxygenation of the anoxic myocardium will, under certain conditions, lead to a resumption of contractile activity. While it
METABOLIC
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has been shown that high concentrations of K+ can induce some degree of enzyme release under aerobic conditions [Z], it has also been demonstrated by the same workers that contraction is necessary for this enzyme release to occur. In order to overcome the possibility of enzyme release induced by such contractile activity it was decided to K+ arrest the hearts for the duration of the experimental periods. For functional recovery studies, following the initial wash-out period, the hearts were converted to atrially perfused working preparations. The hearts were then perfused (standard perfusion fluid plus glucose) aerobically for a 15 min control period. During this time, the stability of the preparation could be confirmed and also control values for the working hearts could be established. For example, as an index of external work, the aortic flow rate against a hydrostatic pressure of 100 cm of water could be monitored. At the end of the control period the preparations were converted to an anoxic K+ arrested non-working Langendorff preparation for a 30 min period. At the end of this period the hearts were simultaneously reoxygenated and converted into working preparations perfused with standard perfusion fluid plus glucose. The recovery of aortic flow was monitored over a 20 min period and was expressed as a percentage of the control value for the pre-anoxic working heart. In this way, the effect of additives to the perfusion fluid during anoxia could be measured. Tissue enzyme extraction
For the determination of the total activity of enzymes in fresh myocardium an extraction procedure based on that described by Sobel et al. [Zq was used. Rat hearts were removed, weighed, and perfused for a 2 min period by a Langendorff procedure using standard buffer plus glucose. The heart was then freeze clamped between stainlesssteel tongs cooled to the temperature of liquid nitrogen [31]. The frozen muscle was then placed in liquid nitrogen and powdered in a percussion mortar. The resulting powder (0.2g/ml) was homogenised in an extracting solution of Tris (hydroxymethyl) aminomethane (0.1 M) containing sucrose (0.25 M), ethylenediaminetetraacetic acid (0.001 M) and mercaptoethanol (0.001 M) at pH 7.4 at 4°C. The homogenate was centrifuged (2000 g, 10 min at 4”C), the pellet was discarded and the supernatant was recentrifuged (10000 g, 15 min at 4°C). The supernatant was retained for analysis. Any intact mitochondria in the pellet were disrupted in extracting solution (in which mercaptoethanol was replaced by sodium deoxycholate 0.5% w/v) pH 8.5 for 30 min and then centrifuged (120000 g for 2 h at 4°C). The supernatant was retained for analysis. Analytical
procedures
The perfusate was analysed for creatine phosphokinase-CPK-(ATP: creatine phosphotransferase E.C.2.7.3.2.) glutamate oxaloacetate transaminase-GOT-
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D. J. HEARSE
AND
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(L-Aspartate: 2-oxoglutarate aminotransferase, E.C.2.6.1.1.), cc-hydroxybutyrate dehydrogenase-HBDH-(D-2-hydroxybutyrate: NAD oxidoreductase) and adenylate kinase-AK-(ATP: AMP phosphotransferase. E.C.2.7.4.3.). All analytical procedures were as described by Hearse et al. [S]
Units of activity
Enzyme activity releasedto the perfusate was expressedasm IU of enzyme activity released/mm or total m IU released/perfusion period. Total enzyme activity recovered from fresh heart was expressedas m IU enzyme activity/heart.
3. Results Enzyme release during anoxia
The releaseof four enzymes (CPK, AK, HBDH and GOT) was measured over a 7 h anoxic period. During anoxia, there was no glucosein the perfusion fluid. The results (Figure 1) indicate extensive enzyme releaseover the 7 h period. The time of initial release, the rate of release and the total amount releasedvaried with the
Time
(mini
the FIGURE 1. Enzyme release profiles (mIU/ min) from the isolated perfused rat heart following onset of anoxia (t=O). (a) cc-hydroxybutyrate dehydrogenase; (0) creatine phosphokinase; (0) For details of perfusion see text. Each adenylate kinase; (m) glutamate oxaloacetate transaminase. point represents the mean for four hearts and the bars represent the S.E.M.
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ANOXIA
individual enzymes. CPK was released first, appearing approximately 15 to 20 min after the onset of anoxia. In all instances enzyme release was biphasic. Phase 1 release usually occurred during the first 20 to 100 min of anoxia and represented 2 to 5% of the total enzyme lost over the 7 h period. During phase 2 release, which lasted several hours, the bulk of the enzyme was released. Despite maintained anoxia, enzyme release declined after approximately 6 h. This may indicate major depletion of the total cellular complement of the various enzymes. This possibility was confirmed by comparing the total enzyme released over the 7 h period with the total enzyme activity recoverable from fresh heart (Table 1). The comparison of tissue enzyme levels (based on the determination of the enzyme that may be extracted into sucrose solution) and perfusate enzyme levels (based on direct determination) is subject to some criticism. Efficient extraction of enzymes from tissue homogenates is not simple to achieve or quantitate, especially as different enzymes will exhibit varying extraction characteristics. Despite this limitation in methodology the amounts of activity extractable from fresh heart were of the same order as the total amounts of activity recovered in the perfusion fluid. TABLE
1. Total enzyme mitochondrial obtained under determinations
activities recovered from fresh heart (combined solubilized and cytoplasmic fraction) and also from coronary perfusate a variety of conditions. Each value is the mean of four separate
Enzyme Condition
HBDH
Fresh heart, mIU/heart 7h anoxia, mIU released 6h anoxia, mIU released 6h anoxia plus glucose, mIU released 6h hypoxia, 23% 0s mIU released 6h hypoxia, 5% 02 mIU released 6h hypoxia, 74% 0~ mIU released 6h hypoxia, 10% 0s mIU released 6h hypoxia, 20% 0s mIU released 6h hypoxia, 10% 0s plus glucose, mIU released 6h anoxia plus methyl prednisolone, mIU released 150 min anoxia plus glucose, 50 min reoxygenation plus glucose mIU released 150 min hypoxia, 10% 02, 50 min reoxygenation, mIU released 150 min anxoia, 50 min reoxygenation, mIU released 150 min auxoia, 50 min reoxygenation plus glucose, mIU released 150 min anoxia, 50 min reoxygenation plus methyl prednisolone mIU released 150 min auxoia, 50 min reoxygenation plus ascorbate, mIU released
118000 181000 134000 13000 102000 86000 43000 19000 18000 1800 60 000
CPK
GOT
AK
292 000 312000 256000 29000 297000 160000 105000 68000 42000 10000 172 000
99 000 77000 65000 9700 30000 37000 12 000 8000 6000 400 24000
2800
14900
1900
8200
1000 2 15000
1900 400000
7700 90000
1300 121000
82000
351000
34000
127000
106000
238000
31000
147000
51000
108000
24000
91000 70000 62 000 3800 55 000 46 000 31000 29000 11000 800 100000
63 000
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D. J. HEARSE
AND
S. M. HUMPHREY
A characteristic feature of the enzyme leakage during anoxia was the biphasic release profile. This biphasic profile was apparent whether the results were expressedas m I U /min or m I U /ml. The causeof this effect is not known, it does not appear to be related to vascular damage associatedwith the mounting procedure as mounting the heart without prior hypothermic arrest, or allowing 30 min aerobic perfusion before anoxia, does not prevent the biphasic release. The use of K+ arrest during anoxia is not responsible for the effect either, as experiments with normal concentrations of Kf during anoxia also produce biphasic release. Some clue to the nature of this effect may lie in the observation that in someof the experiments described below, e.g. glucoseprotection, phase 1 releaseis lost. In addition, reoxygenation during phase 1 [7, 81, in contrast to reoxygenation during phase 2 doesnot induce the exacerbation of release.It may well be that the relatively small enzyme releaseassociatedwith phase 1 arisesfrom a distinct tissue area or type and may not be directly associatedwith the myocardial muscle cell damage. Kubler et al [15] have suggestedthat there is a marked difference between cardiac conducting tissue and cardiac muscle in the susceptibility to hypoxic and ischemic damage.
Protection by glucose
Preliminary experiments [IS] with an isolated working heart preparation using a different experimental time course revealed that the inclusion of glucose in the perfusion fluid during anoxia led to a major reduction in myocardial enzyme release. The enzyme releasestudies, described in the above section, were therefore repeated with exogenous supplies of glucose (11.1 mM) available to the anoxic myocardium. For clarity, Figure 2 shows the resultant releaseprofile for HBDH only but is representative of all enzymes studied. Table 1 showsthe effect of glucose on the total enzyme releasedduring the experimental period for all enzymes studied. The inclusion of glucose leads to a considerable extension of the period of anoxia without significant enzyme releaseand also a major reduction in the overall extent of release (90% reduction for HBDH over 6 h).
Protection by partial
oxygen ~uu~~ubiZi~
Investigations with graded hypoxia were undertaken to determine whether any protective effect could be afforded by a partial availability of oxygen. Hearts were perfused for 6 h with glucose free perfusion fluid that had been equilibrated with a variety of gas mixtures. Figure 3 showsthe results for HBDH and is representative of the other enzymes studied, the effect on cumulative enzyme release for each
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r-
2
9oc )-
E 3 s $j 6OC )al 5 E 2 L5
3oc )-
C
/’ / */t-*1, .-_-/>v--j--~. ‘0
60
120
180
240
300
36C
Time (min 1
FIGURE 2. Profiles for the release of HBDH (mIU/ min) from the isolated perfused rat heart following the onset of anoxia (t=O). (0) anoxic perfusion in the absence of exogenous glucose; (0) anoxic perfusion with glucose ( 11.1 111~) added to the perfusion fluid. Each point represents the mean for four hearts and the bars represent the S.E.M.
enzyme is shown in Table 1. In comparison with the enzyme release observed during total anoxia, with 2+% or 5% 02, there was some reduction in the rate of enzyme release such that over the same time period only 65 to 75% of the anoxic HBDH release was observed. When oxgyen availability was increased to 73% or 10% 02 a further reduction in both the rate and extent of release occurred. In addition there was a considerable delay prior to the onset of release. Increasing the 0s to 20% resulted in a marked shift with little HBDH release for 220 min and also a reduction of release to about 13% of the anoxic control level. Thus for HBDH and the other enzymes studied, increasing the 0s availability up to 20% resulted in a progressive decrease in the extent of release and an increase in the duration of anoxia without release. The protective effects of oxygen and glucose were shown to be additive and in studies with glucose (11.1 mM) and 0s (10%) in the anoxic perfusion fluid there was a further large reduction in the extent of enzyme release (Figure 3).
Protection by methyl prednisolone
The reduction of enzyme releaseresulting from the availability of exogenousglucose or a partial supply of oxygen may possibly occur asa consequenceof the increased energy yielding reactions promoted by these interventions (see discussion). Thus,
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HUMPHREY
Time ( min)
FIGURE 3. Profiles for the release of HBDH rat heart (mIU/ min) from the isolated perfused following the onset ofhypoxia (t=O), (0) 0% oxyg en; (+I 23%owgen; (0) 5% oxygen; (0) 73% oxygen; (0) 10% oxygen; (m) 20% oxygen; (A) 10% oxygen plus glucose (11.1 mu). Each point represents the mean for four hearts.
increased levels of ATP may supply energy for the maintenance of the biochemical and morphological integrity of the cell. The leakage of intracellular enzymes reflects alterations or lesions of cellular membranes, it may be argued therefore that interventions aimed at stabilizing cellular membranes may have some overall protective effect upon the cell. In this connection there is some evidence in the literature [12, 291 for the protective effect of membrane stabilizing durgs such as methyl prednisolone. Hearts were therefore perfused anoxically with glucose free buffer containing methyl prednisolone (0.01 mM). The results obtained were somewhat variable from enzyme to enzyme. Whereas a reduction in release was observed for HBDH (Figuk 4) CPK and GOT, with AK there was an apparent increase in the release. While the reduction of HBDH release was apparent throughout the anoxic period, with CPK and GOT the reduction of release was confined to the earlier stages of release. This observed variability in the reduction of release for the different enzymes in comparison with the more uniform effects observed with glucose or partial oxygen availability questions the meaning of the methyl prednisolone results. Enzyme leakage is only a reflection of major myocardial damage and it becomes important to question whether any reduction of enzyme loss represents a true metabolic protection with increased myocardial viability (as occurs with glucose protection [5, 61) or whether it just prevents enzyme loss without substantially affecting the primary metabolic lesion. The effect of methyl prednisolone on the viability of the anoxic heart was there-
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Time ( min 1
FIGURE 4. Profile following the onset of prednisolone (0.01 mM) and the bars represent
for the release of HBDH rat heart (mIU/ min) from the isolated perfused anoxia (t=O). (e) anoxic perfusion; (0) anoxic perfusion with methyl added to the perfusion fluid. Each point represents the mean for four hearts the S.E.M.
fore investigated. Following an aerobic working control period, hearts (n=6) were subjected to 30 min anoxic Langendorff perfusion with glucose-free perfusion fluid containing 16mM K+. In a further group of six hearts, methyl prednisolone (O.OlmM) was included in the anoxic perfusion fluid and in an additional group (n=6), glucose (11.1 mM) was included. At the end of the anoxic period the hearts were simultaneously reoxygenated and converted to a working preparation perfused with glucose supplemented buffer and the recovery was monitored and expressed as a percentage of the pre-anoxic control value. The results (Figure 5) illustrate that the potassium arrested hearts exhibit a poor recovery and that the recovery profile is not significantly improved by the inclusion of methyl prednisolone in the anoxic perfusion fluid. In contrast the inclusion of glucose considerably improved the recovery profile. These results would be consistent with the observed general reduction in enzyme release and possible metabolic protection with glucose and the variable results obtained with methyl prednisolone. In the latter case, there is no increase in cell viability and any reduction in enzyme release may represent some physical membrane effect. Metabolic The
results
described
protection or delayed release?
so far for glucose
and
oxygen
and
possibly
for methyl
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Recovery
S. M. HUMPHREY
time
(min)
FIGURE 5. The recovery ofaortic ffow rate in the isolated perfused working rat heart after 30 min anoxic perfusion. The composition of the perfusion fluid during anoxia was : (0) 16.0 mx K+, 11.1 mM glucose; (m) 16.0 mu K+, glucose free; (0) 16.0 rnM K+, 0.01 mM methyl prednisolone, glucose free. Each point represents the mean of 6 hearts and the bars represent the S.E.M.
prednisolone show that it is possible to reduce both the rate and extent of anoxic enzyme release. It could be questioned whether the observed reduction represents a true metabolic protection or only a delayed release. Previously we have reported [8] that reoxygenation of the myocardium following the onset of enzyme release leads to no reduction in the extent of release. In fact, after an extended period of anoxia (100 min or more), the readmission of molecular oxygen greatly exacerbates enzyme release. In less than 2 min, the level of enzyme release is increased by as much as 100 fold and the amount of enzyme which would normally be released over 6 h anoxia is released in less than 30 min. Recent studies [9] have shown that this exacerbation of release occurs as a result of oxygen induced extensive ultrastructural damage. The reoxygenation model provides a further means of distinguishing between true metabolic protection and delayed release. Hearts were subjected to reoxygenation after 150 min anoxic perfusion with buffer containing either glucose (11.1 mM) or methyl prednisolone (O.OlmM) or after 150 min hypoxic (10% 02) perfusion. The rest&s (Figure 6 and Table 1) show that with both glucose and 10% 0~ there is complete protection such that following reoxygenation there is no significant exacerbation of enzyme release. In contrast, methyl prednisolone failed to prevent the exacerbation of release. These results provide further evidence that, while
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Time (min)
FIGURE 6. Profile for the release of HBDH (mIU/ min) from the isolated perfused rat heart. Hearts were subjected to reoxygenation for 50 min following 150 min perfusion with (0) anoxia plus 11.1 mM glucose, 16.0 ITIM K+; (m) anoxia plus 0.01 rnM methyl prednisolone, 16.0 mM K+; oxia, 10% 0s 16.0 rnM K+; (0) anoxia, 16.0 rnM K+. Each point represents the mean for
glucose and oxygen are able to confer true metabolic viability, methyl prednisolone is unable to effectively against anoxic damage.
The tranrition
protection with increased protect the myocardium
to irreversible damage and the timing of a metabolic intervention
The results described so far provide evidence that, if initiated in time, metabolic protection is able to improve substantially the status of the anoxic myocardium. In these studies, however, the introduction of the metabolic protection has been coincident with the onset of anoxia and has thus been initiated before the onset of reversible or irreversible damage. Following the onset of irreversible damage it should, by definition, be impossible to reverse damage with any protective agents.
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In view of this and also the proposition [8] that the onset of major enzyme leakage from the anoxic myocardium signals the transition from reversible to irreversible damage, then once initiated, it should not be possible to halt or substantially reduce enzyme release. This possibility was investigated by determining the efficacy of introducing protective agents, in addition to oxygen, to the myocardium after the onset of enzyme release. After 150 min of anoxic substrate free perfusion, hearts were simultaneously reoxygenated and perfused with either normal perfusion fluid, or perfusion fluid plus glucose (11.1 mM), or perfusion fluid plus methyl prednisolone (0.01 mM) or perfusion fluid plus sodium ascorbate (1 .O mM). With reference to the latter condition the possibility exists that the loss of cellular antioxidants (10) e.g. ascorbate may contribute towards the exacerbation of enzyme release induced by reoxygenation. (a)
c
ooo12
I
9000 -
ID *
6000 5 .E
i
0
3000-
s
0
100
I 4
D-0 i 150
I 200
Cc) ;
L
12000-
Cd)
A 9000 -
0
6000 -
0 bI0
3000 -
0I I a ” 1 L7 100 -150 200 Time ( min )
FIGURE 7. Profile for the release of HBDH from the isolated perfused rat heart following the onset of anoxia (t= 0). Hearts were perfused under anoxic conditions in the absence of glucose for 150 min, Hearts were reoxygenated at t=150 min. (a), reoxygenation with perfusion fluid; (b), reoxygenation with perfusion fluid plus methyl prednisolone (0.01 mM); (c), reoxygenation with with perfusion fluid plus sodium ascorbate perfusion fluid plus glucose ( 11.1 mna), (d) , reoxygenation (1 .O 111~). Each point represents the mean for four hearts.
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The results of these studies (Figure 7) indicate that in each case, despite the inclusion of a protective agent, there was a massive increase in enzyme release following reoxygenation. The results are shown for HBDH but are representative of all enzymes studied. In every case the rate of enzyme release was increased between 30 and 100 fold. While there was evidence of some protection in some cases, particularly with ascorbate, due to the remaining magnitude of release, this reduction is likely to be of little practical importance.
The e$ct of oxygen tension upon the extent of exacerbation of enzyme release induced by reoxygenation The reoxygenation studies described in the preceding section involved the sudden exposure of previously anoxic tissue to high oxygen tensions. While there was considerable evidence [8] that the exacerbation of release was directly attributable to the readmission of molecular oxygen, no studies have been made into the relation between the extent of exacerbation and the degree of reoxygenation, nor has the possibility been explored that a gradual or stepwise reoxygenation may possibly circumvent the exacerbation. In preliminary studies hearts were subjected to 150 min anoxia in the absence of exogenous glucose. The hearts were then perfused for successive 5 min periods with perfusion fluid that had been gassed with 30%, 50%, 60% and 80% oxygen. In each instance there was a marked increase in enzyme release. The higher the oxygen tension the greater was the exacerbation. Investigating this further and in an attempt to ascertain whether there was a threshold value below which exacerbation did not occur, experiments were carried out with reoxygenation at a series of different oxygen tensions. Following reoxygenation, the total number of units of enzyme activity released to the perfusate over a 30 min period was determined and was related to the oxygen content of the perfusate. The results (Figure 8) reveal that as the oxygen tension increases so does the extent of exacerbation. For example, in the absence of reoxygenation (POs less than 5 mm Hg), 13000 mIU of HBDH are released in 30 min, reoxygenation with 10% 02 (PO2 approximately 70 mm Hg) increased the release to 19000 mIU/30 min; 20% O2 (POa approximately 140 mm Hg) gave 26000 mIU/30 min; 40% 0s (POs approximately 280 mm Hg) gave 36000 mIU/30 min; 60% 0s (PO2 approximately 430 mm Hg) gave 44000 mIU/30 min; 70% 0s (PO2 approximately 500 mm Hg) gave 57000 mIU/30 min; 80% 0s (PO2 approximately 570 mm Hg) gave 66000 mIU/SO min and 95% 02 (PO2 approximately 680 mm Hg) gave 112000 mIU/30 min. In all experiments at the end of the 30 min period of reoxygenation, the hearts were further reoxygenated with 95% 02. In the case of the OS, lo%, 20x, 40% and 45% 02 this led to an additional exacerbation of release to give a total release similar to that which would have been obtained with a single reoxygenation with
478
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120 000
AND
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HUMPHREY
l-
I I O 0
I 200 PO,
mmHg
I
I
400
600
foweoxygenafion
FIGURE 8. The exacerbation of HBDH release from the isolated perfused anoxic (150 min) rat heart after reoxygenation at different oxygen tensions. Hearts were reoxygenated for 30 min and the total enzyme released (mIU) to the perfusion fluid was determined. For details see text. Each point represents the mean for four hearts and the bars represent the S.E.M.
higher oxygen concentrations. It therefore appears that the extent of exacerbation of enzyme release induced by reoxygenation is dependent upon the concentration of 0s and furthermore it cannot be overcome by a process of gradual reoxygenation.
4. Discussion The possibilities and limitations of metabolic protection of the ischemic myocardium have been discussed for many years. From the early clinical studies of Sodi-Pallares [27] into the use of glucose, potassium and insulin there have been many clinical and experimental studies into possible methods by which the myocardium could be supported during ischemia or anoxia. Many of these studies [8, 12, 22, 301 have further underlined the potential value of metabolic protection. The studies reported in this paper have utilized the anoxic and hypoxic isolated perfused rat heart. It is clearly appreciated [5] that this model differs from the ischemic heart segment in many respects, despite the inadequacies of the model it is
METABOLIC
PROTECTION
DURING
MYOCARDIAL
ANOXIA
479
noteworthy that the characteristic enzyme release profiles observed in these and other studies [I, 61 have features in common with the profiles from ischemic dog and human hearts. For example, creatine phosphokinase activity is detected first and appears at a faster rate and in larger amounts than the other enzymes. a-Hydroxybutyrate dehydrogenase appears later but the release is maintained for a longer period of time. Using enzyme release as an index of myocardial damage the results described in this paper illustrate that with suitable interventions it is possible to considerably modify the pattern of cardiac enzyme release. It is however, important to question whether any reduction in enzyme release represents a true metabolic protection with improved viability or whether enzyme release has only been delayed or prevented, possibly by some physical effect, without enhancing the survival of the cell. The results show that the inclusion of glucose in the perfusion fluid during anoxia increases the duration of anoxia that can be tolerated without enzyme release and also reduces the rate of enzyme release once initiated. Although the inclusion of glucose at the time of reoxygenation does not greatly reduce the exacerbation of release, if glucose is included in the anoxic perfusion fluid for the entire anoxic period the reoxygenation phenomenon is prevented. These results would suggest that glucose exerts a true protective effect. We would suggest that this is a metabolic effect brought about by the ability of glucose to act as an energy source during anoxia. Under similar conditions we have shown [5, 61 that the glucose increases the myocardia1 level of ATP and also the ability of the heart to survive during, and recover from, anoxia. The increased availability of ATP would facilitate an extended period of cellular maintenance and prevent the deterioration which is intensified by reoxygenation. The possibility cannot be excluded that the protective effect of glucose is not entirely metabolic in origin and there may be some contribution from osmotic effects [18]. The results with graded hypoxia were very similar to those with glucose. An increasing availability of oxygen afforded increasing degrees of protection. This protective effect was sufficient to prevent the reoxygenation phenomenon. As with glucose, the limited availability of oxygen would facilitate increased ATP production, this time through a partially operative oxidative metabolism. The effects of glucose and hypoxia were shown to be additive and further increased the protective effect. With methyl prednisolone no clear protective effect was observed, while there was some reduction of enzyme release this was variable and the inclusion of methyl prednisolone during anoxia was not able to prevent the reoxygenation phenomenon. These results coupled with the inability of the compound to increase the functional recovery of the working heart after 30 min anoxia would suggest that methyl prednisolone is unable to improve cell viability and therefore does not offer a true protective effect. However, Libby et al. [19] have reported that hydrocortisone administration, in addition to reducing CPK loss in the infarcting dog heart,
480
D.J.HEARSE
AND S.M.HUMPHREY
also reduces the ST-elevation and the ultrastructural damage associated with myocardial ischemia. The reoxygenation phenomenon appears to represent the compression of damage into a few minutes which would normally have taken several hours. The effect cannot be prevented by the inclusion of protective agents at the time of reoxygenation, the inclusion of antioxidants or the use of gradual reoxygenation. Once the cell has deteriorated beyond a certain point enzyme release cannot be reduced and the reoxygenation phenomenon cannot be prevented. The action of protective agents is to prevent the cell deteriorating to the point where it becomes susceptible to damage. We would suggest that protective agents introduced during the initial phase of the reversible damage [II] will be effective in reducing cellular damage and thus delay the onset of irreversible damage. However once irreversible damage is initiated little can be done to prevent tissue deterioration and the cells become susceptible to effects such as the reoxygenation phenomenon. The absence of a major increase in enzyme release following reoxygenation during phase 1 release [7, 81 may indicate that irreversible damage to the bulk of the myocardium has not occurred and that the occurrence of irreversible damage is represented by the onset of phase 2 release. These results would underline the importance of the need for the rapid introduction of protective agents to the anoxic or hypoxic myocardium. The sooner the introduction after the onset of hypoxia the greater will be the chance of significant metabolic protection. Protection must be initiated before the transition from reversible to irreversible cellular damage and this may perhaps be best achieved by interventions aimed at reducing cellular energy demands and maximizing cellular energy production.
Acknowledgements
This work was carried out with the aid of a grant from the British Heart Foundation. We acknowledge the assistance of Caroline Tahourdin in the methyl prednisolone recovery studies and David Stewart for computer programming.
REFERENCES 1. BUTTERWORTH, P.J., PLUMMER, D. T., ROCHMAN, H. & WEBER, W. V. Factors affecting the release of enzymes from the isolated perfused rat heart. Proceedings of the Physiological Society 3P-4P (1970). 2. DE LEIRIS, J., BRETON, D., FEUVRAY, D. & CORABOEUF, E. Lactic dehydrogenase release from perfused rat heart under the effect of abnormal media. Archives of hternational PhysioloQ and Biochemistry 77, 749-762 (1969). 3. EWEN, L. M. & GRIFFITHS, J. Patterns of enzyme activity following myocardial infarction and ischemia. American Journal of Clinical Pathology 56, 6 14622 (197 1).
METABOLIC 4.
5. 6.
7.
8.
9.
10. 11. 12. 13. 14. 15. 16. 17. 18.
19.
PROTECTION DURING MYOCARDIALANOXIA
481
GUDBJARNASON, S., MATHES, P. & RAVENS, K. G. Functional compartmentation of ATP and creatine phosphate in heart muscle. Journal of Molecular and Cellular Cardiology 1, 325-339 (1969). HEARSE, D. J. & CHAIN, E. B. The role of glucose in the survival and recovery of the anoxic isolated perfused rat heart. Biochemical Journal 128, 1125-l 133 (1972). HEARSE, D. J. & CHAIN, E, B. The effect of glucose on enzyme release from, and recovery of, the anoxic myocardium. In Myocardial Metabolism: Recent Advances in Studies on Cardiac Structure and Metabolism. Vol. 3, 763-772. Baltimore: University Park Press (1973). HEARSE, D. J., HUMPHREY, S. M. & CHAIN, E. B. The effect of reoxygenation on enzyme release from the anoxic isolated perfused rat heart. Biochemical Society Transactions 1, 869-871 (1973). HEARSE, D. J., HUMPHREY, S. M. & CHAIN, E. B. Abrupt reoxygenation of the anoxic potassium-arrested perfused rat heart: A study of myocardial enzyme release. Journal of Molecular and Cellular Cardiology 5, 395407 (1973). HEARSE, D.J., HUMPHREY, S. M., NAYLER, W. G., SLADE, A. & BORDER, D. Ultrastructural damage associated with reoxygenation of the anoxic myocardium. Journal of Molecular and Cellular Cardiology 7, 3 15-324 ( 1975). JAMIESON, D. & VAN DEN BRENK, H. A. S. The effects of antioxidants on high pressure oxygen toxicity. Biochemical Pharmacology 13, 159-164 (1964). JENNINGS, R. B., SOMMERS, H. M., HERDSON, P. B. & KALTENBACH, J. P. Ischemic injury of myocardium. Annals of the New York Academy of Sciences 156, 61-78 (1969). JEWITT, D. E., &ELTON, C. L. & SONNENBLICK, E. H. Salutary effect of methyl prednisolone on the viability of heart muscle exposed to anoxia. Circulation XLVI, 121 (1972). KJEKSHUS, J. K. & SOBEL, B. E. Depressed myocardial creatine phosphokinase activity following experimental myocardial infarction in rabbit. Circulation Research 27, 403414 (1970). KREBS, H. A. & HENSELEIT, K. Untersuchungen iiber die Harnstoffbildung im Tierkijrper. Hoppe-Seyler’s zeitschrift ftir physiologsche Chemie 210, 33-66 ( 1932). K~~BLER, W., MOLL, W., ROGGENDORF, H. & VON SMEKAL, P. Comparative studies of the energy metabolism of the conductive tissue and of the myocardium. In Les Surcharges Cardiaques. P. Y. Hatt Ed. pp. 139-152. Paris: Inserm (1972). K&LER, W. & SPIECKERMANN, P. G. Regulation of glycolysis in the ischemic and anoxic myocardium. Journal of Molecular and Cellular Cardiology 1, 351-377 (1970). LANGENDORFF, 0. Untersuchungen am uberlebenden Saugethierherzen. Pjcger’s Archiv fur die gesamte Physiologie des Men&en und der Tiere 61, 291-332 (1895). LEAF, A. Cell Swelling. Circulation XLVIII, 455-458 (1973). LIBBY, P., MAROKO, P.R., BLOOR,~. M., SOBEL, B.E. &BRAUNWAL.D, E.Reduction of experimental myocardial infarct size by corticosteroid administration. Journal of Clinical
Investigation
52, 599-607
(1973).
NEELY, J. R., LIEBERMEISTER, H., BATTERSBY, E. J. & MORGAN, H. E. Effect of pressure development on oxygen consumption by the isolated rat heart. American Journal of Physiol+p 212, 804-814 (1967). 2 1. NEELY, J. R.,ROVETTO, M. J., WHITMER, J. T. & MORGAN, H. E. Effects of&hernia on function and metabolism of the isolated working rat heart. American 3ournaE of Physiology 225, 651-658 (1973). 22. OPIE, L. H. The glucose hypothesis; Relation to acute myocardial ischemia. Journal of Molecular and Cellular Cardiology 1, 107-l 15 ( 1970). 23. SHELL, W. E.,KJEKSHUS,J. K. & SOBEL,B. E. Quantitative assessmentoftheextentof 20.
482
24. 25. 26.
27. 28. 29.
30.
31.
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AND S.M. HUMPHREY
myocardial infarction in the conscious dog by means of analysis of serial changes in serum creatine phosphokinase activity. Journal of Clinical Investigation 50, 2614-2625 (1971). SOBEL, B. E. Biochemical and morphologic changes in infarcting myocardium. Hospital Practice, 59-7 1 ( 1972). SOBEL, B. E. & SHELL, W. E. Serum enzyme determinations in the diagnosis and assessment of myocardial infarction. Circulation XLV, 471482 (1972). SOBEL, B. E., SHELL, W. E. & KLEIN, M. S. An isoenzyme of creatine phosphokinaes associated with rabbit heart mitochondria. Journal of Molecular and Cellular Cardiology 4, 367-380 (1972). SODI-PALLARES, D. The importance of electrolyte therapy in heart disease, Annals ofthe Jvezv York Academy of Sciences 156, 603-619 (1969). UMBREIT, W. W., BURRIS, R. H. & STAUFFER, J. F. In Manomtric Techniques, pp. 132, Minneapolis : Burgess ( 1964). WAGNER, J. A. & CRITZ, J. B. The effect of prednisolone on the serum creatine phosphokinase response to exercise. Proceedings of the Society for Experimental Biology and Medicine 128, 716-720 (1968). WEISSLER, A. M., KRUGER, F. A., BABA, N., SCARPELLI, D. G., LEIGHTON, R. F. & GALLIMORE, J. K. Role of anaerobic metabolism in the preservation of functional capacity and structure of anoxic myocardium. Journal of Clinical Investigation 47, 403416 (1968). WOLLENBERGER, A., RISTAU, 0. & SCHOFFA, G. Eine einfache Technik der extremschnellen Abkiihlung grosserer Gewebestucke. Pjggers Archiv ftir die Gesamte Physiologic des Men&en und der Tiere 270, 399-412 (1960).
of Motecatur
and Cellular
Enzyme
Def>artment
Cardiology
(1975)
7,463-482
Release During Myocardial Anoxia A Study of Metabolic Protection
D. J. HEARSE AND S. M. HUMPHREY of Biochemistry, Imfierial College of Science, Kensington,
(Received
26 October
1973,
accebted in revisedform
24 June
:
London,
U.K.
1974)
D. J. HEARSE AND S. M. HUMPHREY. Enzyme Release During Myocardial Anoxia: A Study of Metabolic Protection. Journal of Mole&r and Cellular Cardiology (1975) 7, 46% 482. In studies with the isolated perfused anoxic rat heart, myocardial enzyme release was used as an index of cellular damage. Anoxic damage (with K+ arrest) was characterized by measuring the release of four enzymes (creatine phosphokinase, a-hydroxybutyrate dehydrogenase, glutamate oxaloacetate transaminase and adenylate kinase). The possibility of protecting the myocardium against anoxic damage by using potential metabolic protective agents was investigated. The inclusion of glucose as an anaerobic energy source in the perfusion fluid during the entire anoxic period afforded considerable metabolic protection, the period of anoxia that could be tolerated without significant enzyme release was extended and the overall release was reduced. In addition, glucose during anoxia was able to confer protection against the exacerbation of enzyme release induced by reoxygenation. The results with graded hypoxia were similar to those with glucose and the two were additive. An increasing availability of oxygen afforded an increasing degree of protection. The membrane stabilizing drug methyl prednisolone had a variable effect upon enzyme release but was unable to confer true protection with increased viability upon the myocardium. The exacerbation of enzyme release induced by reoxygenation was further investigated. Potential protective agents (glucose, methyl prednisolone and the anti-oxidant ascorbate) introduced at the time of reoxygenation had little protective effect. Stepwise reoxygenation studies revealed that the extent ofexacerbation was determined by the concentration of 0s used in the reoxygenation process, the higher the 0s tension the greater the exacerbation of release. It is proposed that the onset of major myocardial enzyme release reflects the transition from reversible to irreversible cellular damage and that the action of metabolic protective agents is to prevent the deterioration of the cells to a point where they become susceptible to irreversible damage. Once the cells enter a state of irreversible damage and major enzyme release occurs then metabolic protection becomes ineffective. The results underline the importance of the need for the rapid introduction of protective agents to the anoxic myocardium. In this experimental model, protection must be initiated before the transition from reversible to irreversible cellular damage and this may possibly be best achieved either by reducing cellular energy demands or by maximizing cellular energy production. KEY WORDS: Anoxia; Hypoxia; Metabolic protection; Glucose; K+ arrest; Enzyme release; Isolated perfused rat heart; Ascorbate; Methyl prednisolone; Reoxygenation; Creatine phosphokinase; Glutamate oxaloacetate transaminase; Adenylate kinase (myokinase); cr-hydroxybutyrate dehydrogenase; Reversible and irreversible damage.
1. Introduction The appearance in the serum of specific cardiac enzymes has long been used in the diagnosis and assessment of myocardial infarction [3, 251. The leakage of enzymes
464
D.
J.
HEARSE
AND
S. M.
HUMPHREY
from the cell has been associated with biochemical and morphological damage and the extent of enzyme release is related to the severity of cellular damage. From studies of myocardial enzyme depletion in experimentally infarcted dogs it has been suggested [13,23,24] that enzyme release may be used to accurately quantitate the extent of cellular damage. Therapeutic interventions designed to protect the myocardium against damage would be expected to reduce or delay enzyme release and conversely, any extension of damage should lead to an increase in enzyme release. When cellular anoxia, hypoxia or ischemia is suspected, the aim of any intervention is to prevent the onset of irreversible cellular damage. Under conditions of cellular oxygen deprivation, myocardial metabolism is disrupted and despite the activation of anaerobic pathways, cellular energy production is severely impaired. From studies with a variety of experimental models it has been shown that following the onset of anoxia or ischemia myocardial ATP levels decline [4,5, IS, 211 and the cells enter a state of negative energy balance. The availability of adequate supplies of ATP is important not only for contractile activity but also for the maintenance of cellular integrity. It has been suggested [S, 161 that the supply of ATP is a major determinant of cellular survival and recovery and that once the level of myocardial ATP drops below a certain critical level the heart becomes irreversibly damaged and recovery is no longer possible. Associated with the critical reduction of ATP and onset of irreversible damage is the release of enzymes from the myocardium [S]. Effective metabolic protection, preventing or delaying the onset of irreversible damage may possibly be achieved with interventions specifically aimed at maintaining cellular supplies of ATP (for example by providing exogenous glucose supplies for glycolytic ATP production, or by promoting partial oxidative metabolism of endogenous substrates with a limited supply of oxygen). An alternative approach would be to attempt to prevent the loss of cellular enzymes and metabolites by the use of membrane stabilizing drugs, for example methyl prednisolone. The objective of the studies described in this paper was to assess the feasibility of preventing or delaying the onset of anoxia-induced cehular damage with the use of the potential metabolic protective agents described above. In these studies, the release of several enzymes from the isolated perfused rat heart was used as an index of the onset and the severity of myocardial damage. In the light of our recent findings [a] that under certain circumstances reoxygenation of the severely anoxic myocardium can greatly exacerbate enzyme release, we have also investigated the efficacy of these and other potential protective agents (for example gradual reoxygenation or the use of an antioxidant such as ascorbate) in preventing or reducing the oxygen-induced exacerbation of enzyme release. 2. Materials Male
rats (280 to 320g body
and
Methods
wt) of the Sprague-Dawley
strain,
maintained
on a
METABOLIC
PROTECTION
DURING
MYOCARDIAL
ANOXL4
465
standard diet, were used throughout these experiments. All substrates and enzymes used in the analysis were obtained from the Boehringer Corporation (London) Ltd. Methyl prednisolone was generously donated by the Upjohn Company (U.K.) Ltd.
Perfusion techniques Rats were lightly anaesthetized with diethyl ether, the left femoral vein was exposed and heparin (200 IU) was administered intravenously. One min after the administration of heparin, the heart was excised [20] and placed in ice cold perfusion medium until contraction had ceased. The heart was then mounted on the perfusion apparatus. The mounting procedure and apparatus were based on modifications [5] of that described by Neely et al. [20]. The perfusion apparatus was designed so that it could be used for two modes of perfusion which could be readily interconverted.
Non-working
Langendor$peparation
[ 171
Hearts were perfused via the aorta at a perfusion pressure of 100 cm of water. This preparation was used for all enzyme release studies and for the anoxic period in the functional recovery studies.
Working preparation Here hearts were perfused via the left atrium as described by Neely et al. [20] at an atria1 filling pressure of 20 cm of water. The left ventricle spontaneously ejected 40 to 50ml of perfusate/min against a hydrostatic pressure of 100 cm of water. The aortic flow and coronary flow were pooled and recirculated.
Perfusion medium Krebs-Henseleit bicarbonate buffer [14] pH 7.4 was the standard perfusion fluid. In some experiments glucose (11.1 mM) or methyl prednisolone (0.01 mM) or sodium ascorbate (1 .O mM) was included in the buffer. During K+ arrest, the concentration of K+ in the buffer was increased to 16 mM. In high K+ and also sodium ascorbate buffers the concentration of Naf was correspondingly decreased. Precautions [28] were taken to prevent the precipitation of Gas+. Before use, the perfusion fluid was filtered through a cellulose acetate filter of pore size 5.0 km
466
D. J. HEARSE
AND
S. M. HUMPHREY
(Millipore Ltd.). The perfusion fluid was maintained at 37°C and in aerobic or reoxygenation studies the fluid was equilibrated with 02+COz (95:5, aortic 02 partial pressure was over 600 mm Hg), in anoxic studies the fluid was equilibrated with Na+COa (95 :5, aortic 02 partial pressure was less than 5 mm Hg). In hypoxic studies or gradual reoxygenation studies the fluid was equilibrated with N2+02+ CO2, CO2 was maintained at a constant level of 5% while the relative proportions of 02 and N2 were varied to suit the experimental requirement. Atmospheric gas contamination of the heart was prevented by completely enclosing the heart in a waterjacketed (37°C) c h am b er which was continually gassed (20 ml/min) with the same gas mixture as that used for the perfusion fluid.
02 partial pressure measurement This was measured using 02 electrodes (type E5046; Radiometer, Copenhagen, Denmark). 02 partial pressure was continuously monitored using a digital acidbase analyser (type PHM72 ; Radiometer).
Tim8 sequenceof perfwion Immediately after mounting, the heart was perfused (standard perfusion fluid plus glucose at a perfusion pressure of 100 cm of water) aerobically as a non-working Langendorff preparation for a 5 min wash-out and equilibration period. For enzyme release studies, the hearts were maintained as non-working Langendorff preparations for the duration of the experiment. By utilizing a second pregassed perfusion fluid reservoir equipped with a constant head device, the hearts were then subjected to experimental perfusion periods of up to 7 h. In reoxygenation studies additional reservoirs were available for the introduction of suitably gassed perfusion fluid. During the entire experimental time course the coronary flow was recorded and samples of the coronary effluent were taken at 5 min intervals. Immediately after sampling the aliquots were assayed or were stored at 4°C for the duration of the perfusion period. During the enzyme release studies it was decided to potassium arrest the hearts for the duration of the experimental period. There were two reasons for this decision. Firstly, anoxia and hypoxia alone would rapidly induce cardiac arrest, however, arrest would be incomplete and irregular bursts of contractile activity would occur. In addition to making it difficult to standardize perfusion conditions, these anoxic bursts of activity have been associated with some enzyme release (D. J. Hearse and S. M. Humphrey, unpublished results). Secondly, in reoxygenation studies in the absence of K+ arrest, reoxygenation of the anoxic myocardium will, under certain conditions, lead to a resumption of contractile activity. While it
METABOLIC
PROTECTION
DURING
MYOCARDIAL
ANOXIA
467
has been shown that high concentrations of K+ can induce some degree of enzyme release under aerobic conditions [Z], it has also been demonstrated by the same workers that contraction is necessary for this enzyme release to occur. In order to overcome the possibility of enzyme release induced by such contractile activity it was decided to K+ arrest the hearts for the duration of the experimental periods. For functional recovery studies, following the initial wash-out period, the hearts were converted to atrially perfused working preparations. The hearts were then perfused (standard perfusion fluid plus glucose) aerobically for a 15 min control period. During this time, the stability of the preparation could be confirmed and also control values for the working hearts could be established. For example, as an index of external work, the aortic flow rate against a hydrostatic pressure of 100 cm of water could be monitored. At the end of the control period the preparations were converted to an anoxic K+ arrested non-working Langendorff preparation for a 30 min period. At the end of this period the hearts were simultaneously reoxygenated and converted into working preparations perfused with standard perfusion fluid plus glucose. The recovery of aortic flow was monitored over a 20 min period and was expressed as a percentage of the control value for the pre-anoxic working heart. In this way, the effect of additives to the perfusion fluid during anoxia could be measured. Tissue enzyme extraction
For the determination of the total activity of enzymes in fresh myocardium an extraction procedure based on that described by Sobel et al. [Zq was used. Rat hearts were removed, weighed, and perfused for a 2 min period by a Langendorff procedure using standard buffer plus glucose. The heart was then freeze clamped between stainlesssteel tongs cooled to the temperature of liquid nitrogen [31]. The frozen muscle was then placed in liquid nitrogen and powdered in a percussion mortar. The resulting powder (0.2g/ml) was homogenised in an extracting solution of Tris (hydroxymethyl) aminomethane (0.1 M) containing sucrose (0.25 M), ethylenediaminetetraacetic acid (0.001 M) and mercaptoethanol (0.001 M) at pH 7.4 at 4°C. The homogenate was centrifuged (2000 g, 10 min at 4”C), the pellet was discarded and the supernatant was recentrifuged (10000 g, 15 min at 4°C). The supernatant was retained for analysis. Any intact mitochondria in the pellet were disrupted in extracting solution (in which mercaptoethanol was replaced by sodium deoxycholate 0.5% w/v) pH 8.5 for 30 min and then centrifuged (120000 g for 2 h at 4°C). The supernatant was retained for analysis. Analytical
procedures
The perfusate was analysed for creatine phosphokinase-CPK-(ATP: creatine phosphotransferase E.C.2.7.3.2.) glutamate oxaloacetate transaminase-GOT-
468
D. J. HEARSE
AND
S. M. HUMPHREY
(L-Aspartate: 2-oxoglutarate aminotransferase, E.C.2.6.1.1.), cc-hydroxybutyrate dehydrogenase-HBDH-(D-2-hydroxybutyrate: NAD oxidoreductase) and adenylate kinase-AK-(ATP: AMP phosphotransferase. E.C.2.7.4.3.). All analytical procedures were as described by Hearse et al. [S]
Units of activity
Enzyme activity releasedto the perfusate was expressedasm IU of enzyme activity released/mm or total m IU released/perfusion period. Total enzyme activity recovered from fresh heart was expressedas m IU enzyme activity/heart.
3. Results Enzyme release during anoxia
The releaseof four enzymes (CPK, AK, HBDH and GOT) was measured over a 7 h anoxic period. During anoxia, there was no glucosein the perfusion fluid. The results (Figure 1) indicate extensive enzyme releaseover the 7 h period. The time of initial release, the rate of release and the total amount releasedvaried with the
Time
(mini
the FIGURE 1. Enzyme release profiles (mIU/ min) from the isolated perfused rat heart following onset of anoxia (t=O). (a) cc-hydroxybutyrate dehydrogenase; (0) creatine phosphokinase; (0) For details of perfusion see text. Each adenylate kinase; (m) glutamate oxaloacetate transaminase. point represents the mean for four hearts and the bars represent the S.E.M.
METABOLIC
PROTECTION
DURING
MYOCARDIAL
469
ANOXIA
individual enzymes. CPK was released first, appearing approximately 15 to 20 min after the onset of anoxia. In all instances enzyme release was biphasic. Phase 1 release usually occurred during the first 20 to 100 min of anoxia and represented 2 to 5% of the total enzyme lost over the 7 h period. During phase 2 release, which lasted several hours, the bulk of the enzyme was released. Despite maintained anoxia, enzyme release declined after approximately 6 h. This may indicate major depletion of the total cellular complement of the various enzymes. This possibility was confirmed by comparing the total enzyme released over the 7 h period with the total enzyme activity recoverable from fresh heart (Table 1). The comparison of tissue enzyme levels (based on the determination of the enzyme that may be extracted into sucrose solution) and perfusate enzyme levels (based on direct determination) is subject to some criticism. Efficient extraction of enzymes from tissue homogenates is not simple to achieve or quantitate, especially as different enzymes will exhibit varying extraction characteristics. Despite this limitation in methodology the amounts of activity extractable from fresh heart were of the same order as the total amounts of activity recovered in the perfusion fluid. TABLE
1. Total enzyme mitochondrial obtained under determinations
activities recovered from fresh heart (combined solubilized and cytoplasmic fraction) and also from coronary perfusate a variety of conditions. Each value is the mean of four separate
Enzyme Condition
HBDH
Fresh heart, mIU/heart 7h anoxia, mIU released 6h anoxia, mIU released 6h anoxia plus glucose, mIU released 6h hypoxia, 23% 0s mIU released 6h hypoxia, 5% 02 mIU released 6h hypoxia, 74% 0~ mIU released 6h hypoxia, 10% 0s mIU released 6h hypoxia, 20% 0s mIU released 6h hypoxia, 10% 0s plus glucose, mIU released 6h anoxia plus methyl prednisolone, mIU released 150 min anoxia plus glucose, 50 min reoxygenation plus glucose mIU released 150 min hypoxia, 10% 02, 50 min reoxygenation, mIU released 150 min anxoia, 50 min reoxygenation, mIU released 150 min auxoia, 50 min reoxygenation plus glucose, mIU released 150 min anoxia, 50 min reoxygenation plus methyl prednisolone mIU released 150 min auxoia, 50 min reoxygenation plus ascorbate, mIU released
118000 181000 134000 13000 102000 86000 43000 19000 18000 1800 60 000
CPK
GOT
AK
292 000 312000 256000 29000 297000 160000 105000 68000 42000 10000 172 000
99 000 77000 65000 9700 30000 37000 12 000 8000 6000 400 24000
2800
14900
1900
8200
1000 2 15000
1900 400000
7700 90000
1300 121000
82000
351000
34000
127000
106000
238000
31000
147000
51000
108000
24000
91000 70000 62 000 3800 55 000 46 000 31000 29000 11000 800 100000
63 000
470
D. J. HEARSE
AND
S. M. HUMPHREY
A characteristic feature of the enzyme leakage during anoxia was the biphasic release profile. This biphasic profile was apparent whether the results were expressedas m I U /min or m I U /ml. The causeof this effect is not known, it does not appear to be related to vascular damage associatedwith the mounting procedure as mounting the heart without prior hypothermic arrest, or allowing 30 min aerobic perfusion before anoxia, does not prevent the biphasic release. The use of K+ arrest during anoxia is not responsible for the effect either, as experiments with normal concentrations of Kf during anoxia also produce biphasic release. Some clue to the nature of this effect may lie in the observation that in someof the experiments described below, e.g. glucoseprotection, phase 1 releaseis lost. In addition, reoxygenation during phase 1 [7, 81, in contrast to reoxygenation during phase 2 doesnot induce the exacerbation of release.It may well be that the relatively small enzyme releaseassociatedwith phase 1 arisesfrom a distinct tissue area or type and may not be directly associatedwith the myocardial muscle cell damage. Kubler et al [15] have suggestedthat there is a marked difference between cardiac conducting tissue and cardiac muscle in the susceptibility to hypoxic and ischemic damage.
Protection by glucose
Preliminary experiments [IS] with an isolated working heart preparation using a different experimental time course revealed that the inclusion of glucose in the perfusion fluid during anoxia led to a major reduction in myocardial enzyme release. The enzyme releasestudies, described in the above section, were therefore repeated with exogenous supplies of glucose (11.1 mM) available to the anoxic myocardium. For clarity, Figure 2 shows the resultant releaseprofile for HBDH only but is representative of all enzymes studied. Table 1 showsthe effect of glucose on the total enzyme releasedduring the experimental period for all enzymes studied. The inclusion of glucose leads to a considerable extension of the period of anoxia without significant enzyme releaseand also a major reduction in the overall extent of release (90% reduction for HBDH over 6 h).
Protection by partial
oxygen ~uu~~ubiZi~
Investigations with graded hypoxia were undertaken to determine whether any protective effect could be afforded by a partial availability of oxygen. Hearts were perfused for 6 h with glucose free perfusion fluid that had been equilibrated with a variety of gas mixtures. Figure 3 showsthe results for HBDH and is representative of the other enzymes studied, the effect on cumulative enzyme release for each
METABOLIC
PROTECTION
DURING
MYOCARDL4L
471
ANOXIA
r-
2
9oc )-
E 3 s $j 6OC )al 5 E 2 L5
3oc )-
C
/’ / */t-*1, .-_-/>v--j--~. ‘0
60
120
180
240
300
36C
Time (min 1
FIGURE 2. Profiles for the release of HBDH (mIU/ min) from the isolated perfused rat heart following the onset of anoxia (t=O). (0) anoxic perfusion in the absence of exogenous glucose; (0) anoxic perfusion with glucose ( 11.1 111~) added to the perfusion fluid. Each point represents the mean for four hearts and the bars represent the S.E.M.
enzyme is shown in Table 1. In comparison with the enzyme release observed during total anoxia, with 2+% or 5% 02, there was some reduction in the rate of enzyme release such that over the same time period only 65 to 75% of the anoxic HBDH release was observed. When oxgyen availability was increased to 73% or 10% 02 a further reduction in both the rate and extent of release occurred. In addition there was a considerable delay prior to the onset of release. Increasing the 0s to 20% resulted in a marked shift with little HBDH release for 220 min and also a reduction of release to about 13% of the anoxic control level. Thus for HBDH and the other enzymes studied, increasing the 0s availability up to 20% resulted in a progressive decrease in the extent of release and an increase in the duration of anoxia without release. The protective effects of oxygen and glucose were shown to be additive and in studies with glucose (11.1 mM) and 0s (10%) in the anoxic perfusion fluid there was a further large reduction in the extent of enzyme release (Figure 3).
Protection by methyl prednisolone
The reduction of enzyme releaseresulting from the availability of exogenousglucose or a partial supply of oxygen may possibly occur asa consequenceof the increased energy yielding reactions promoted by these interventions (see discussion). Thus,
472
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Time ( min)
FIGURE 3. Profiles for the release of HBDH rat heart (mIU/ min) from the isolated perfused following the onset ofhypoxia (t=O), (0) 0% oxyg en; (+I 23%owgen; (0) 5% oxygen; (0) 73% oxygen; (0) 10% oxygen; (m) 20% oxygen; (A) 10% oxygen plus glucose (11.1 mu). Each point represents the mean for four hearts.
increased levels of ATP may supply energy for the maintenance of the biochemical and morphological integrity of the cell. The leakage of intracellular enzymes reflects alterations or lesions of cellular membranes, it may be argued therefore that interventions aimed at stabilizing cellular membranes may have some overall protective effect upon the cell. In this connection there is some evidence in the literature [12, 291 for the protective effect of membrane stabilizing durgs such as methyl prednisolone. Hearts were therefore perfused anoxically with glucose free buffer containing methyl prednisolone (0.01 mM). The results obtained were somewhat variable from enzyme to enzyme. Whereas a reduction in release was observed for HBDH (Figuk 4) CPK and GOT, with AK there was an apparent increase in the release. While the reduction of HBDH release was apparent throughout the anoxic period, with CPK and GOT the reduction of release was confined to the earlier stages of release. This observed variability in the reduction of release for the different enzymes in comparison with the more uniform effects observed with glucose or partial oxygen availability questions the meaning of the methyl prednisolone results. Enzyme leakage is only a reflection of major myocardial damage and it becomes important to question whether any reduction of enzyme loss represents a true metabolic protection with increased myocardial viability (as occurs with glucose protection [5, 61) or whether it just prevents enzyme loss without substantially affecting the primary metabolic lesion. The effect of methyl prednisolone on the viability of the anoxic heart was there-
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Time ( min 1
FIGURE 4. Profile following the onset of prednisolone (0.01 mM) and the bars represent
for the release of HBDH rat heart (mIU/ min) from the isolated perfused anoxia (t=O). (e) anoxic perfusion; (0) anoxic perfusion with methyl added to the perfusion fluid. Each point represents the mean for four hearts the S.E.M.
fore investigated. Following an aerobic working control period, hearts (n=6) were subjected to 30 min anoxic Langendorff perfusion with glucose-free perfusion fluid containing 16mM K+. In a further group of six hearts, methyl prednisolone (O.OlmM) was included in the anoxic perfusion fluid and in an additional group (n=6), glucose (11.1 mM) was included. At the end of the anoxic period the hearts were simultaneously reoxygenated and converted to a working preparation perfused with glucose supplemented buffer and the recovery was monitored and expressed as a percentage of the pre-anoxic control value. The results (Figure 5) illustrate that the potassium arrested hearts exhibit a poor recovery and that the recovery profile is not significantly improved by the inclusion of methyl prednisolone in the anoxic perfusion fluid. In contrast the inclusion of glucose considerably improved the recovery profile. These results would be consistent with the observed general reduction in enzyme release and possible metabolic protection with glucose and the variable results obtained with methyl prednisolone. In the latter case, there is no increase in cell viability and any reduction in enzyme release may represent some physical membrane effect. Metabolic The
results
described
protection or delayed release?
so far for glucose
and
oxygen
and
possibly
for methyl
474
D. J. HEARSE
AND
Recovery
S. M. HUMPHREY
time
(min)
FIGURE 5. The recovery ofaortic ffow rate in the isolated perfused working rat heart after 30 min anoxic perfusion. The composition of the perfusion fluid during anoxia was : (0) 16.0 mx K+, 11.1 mM glucose; (m) 16.0 mu K+, glucose free; (0) 16.0 rnM K+, 0.01 mM methyl prednisolone, glucose free. Each point represents the mean of 6 hearts and the bars represent the S.E.M.
prednisolone show that it is possible to reduce both the rate and extent of anoxic enzyme release. It could be questioned whether the observed reduction represents a true metabolic protection or only a delayed release. Previously we have reported [8] that reoxygenation of the myocardium following the onset of enzyme release leads to no reduction in the extent of release. In fact, after an extended period of anoxia (100 min or more), the readmission of molecular oxygen greatly exacerbates enzyme release. In less than 2 min, the level of enzyme release is increased by as much as 100 fold and the amount of enzyme which would normally be released over 6 h anoxia is released in less than 30 min. Recent studies [9] have shown that this exacerbation of release occurs as a result of oxygen induced extensive ultrastructural damage. The reoxygenation model provides a further means of distinguishing between true metabolic protection and delayed release. Hearts were subjected to reoxygenation after 150 min anoxic perfusion with buffer containing either glucose (11.1 mM) or methyl prednisolone (O.OlmM) or after 150 min hypoxic (10% 02) perfusion. The rest&s (Figure 6 and Table 1) show that with both glucose and 10% 0~ there is complete protection such that following reoxygenation there is no significant exacerbation of enzyme release. In contrast, methyl prednisolone failed to prevent the exacerbation of release. These results provide further evidence that, while
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475
Time (min)
FIGURE 6. Profile for the release of HBDH (mIU/ min) from the isolated perfused rat heart. Hearts were subjected to reoxygenation for 50 min following 150 min perfusion with (0) anoxia plus 11.1 mM glucose, 16.0 ITIM K+; (m) anoxia plus 0.01 rnM methyl prednisolone, 16.0 mM K+; oxia, 10% 0s 16.0 rnM K+; (0) anoxia, 16.0 rnM K+. Each point represents the mean for
glucose and oxygen are able to confer true metabolic viability, methyl prednisolone is unable to effectively against anoxic damage.
The tranrition
protection with increased protect the myocardium
to irreversible damage and the timing of a metabolic intervention
The results described so far provide evidence that, if initiated in time, metabolic protection is able to improve substantially the status of the anoxic myocardium. In these studies, however, the introduction of the metabolic protection has been coincident with the onset of anoxia and has thus been initiated before the onset of reversible or irreversible damage. Following the onset of irreversible damage it should, by definition, be impossible to reverse damage with any protective agents.
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In view of this and also the proposition [8] that the onset of major enzyme leakage from the anoxic myocardium signals the transition from reversible to irreversible damage, then once initiated, it should not be possible to halt or substantially reduce enzyme release. This possibility was investigated by determining the efficacy of introducing protective agents, in addition to oxygen, to the myocardium after the onset of enzyme release. After 150 min of anoxic substrate free perfusion, hearts were simultaneously reoxygenated and perfused with either normal perfusion fluid, or perfusion fluid plus glucose (11.1 mM), or perfusion fluid plus methyl prednisolone (0.01 mM) or perfusion fluid plus sodium ascorbate (1 .O mM). With reference to the latter condition the possibility exists that the loss of cellular antioxidants (10) e.g. ascorbate may contribute towards the exacerbation of enzyme release induced by reoxygenation. (a)
c
ooo12
I
9000 -
ID *
6000 5 .E
i
0
3000-
s
0
100
I 4
D-0 i 150
I 200
Cc) ;
L
12000-
Cd)
A 9000 -
0
6000 -
0 bI0
3000 -
0I I a ” 1 L7 100 -150 200 Time ( min )
FIGURE 7. Profile for the release of HBDH from the isolated perfused rat heart following the onset of anoxia (t= 0). Hearts were perfused under anoxic conditions in the absence of glucose for 150 min, Hearts were reoxygenated at t=150 min. (a), reoxygenation with perfusion fluid; (b), reoxygenation with perfusion fluid plus methyl prednisolone (0.01 mM); (c), reoxygenation with with perfusion fluid plus sodium ascorbate perfusion fluid plus glucose ( 11.1 mna), (d) , reoxygenation (1 .O 111~). Each point represents the mean for four hearts.
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The results of these studies (Figure 7) indicate that in each case, despite the inclusion of a protective agent, there was a massive increase in enzyme release following reoxygenation. The results are shown for HBDH but are representative of all enzymes studied. In every case the rate of enzyme release was increased between 30 and 100 fold. While there was evidence of some protection in some cases, particularly with ascorbate, due to the remaining magnitude of release, this reduction is likely to be of little practical importance.
The e$ct of oxygen tension upon the extent of exacerbation of enzyme release induced by reoxygenation The reoxygenation studies described in the preceding section involved the sudden exposure of previously anoxic tissue to high oxygen tensions. While there was considerable evidence [8] that the exacerbation of release was directly attributable to the readmission of molecular oxygen, no studies have been made into the relation between the extent of exacerbation and the degree of reoxygenation, nor has the possibility been explored that a gradual or stepwise reoxygenation may possibly circumvent the exacerbation. In preliminary studies hearts were subjected to 150 min anoxia in the absence of exogenous glucose. The hearts were then perfused for successive 5 min periods with perfusion fluid that had been gassed with 30%, 50%, 60% and 80% oxygen. In each instance there was a marked increase in enzyme release. The higher the oxygen tension the greater was the exacerbation. Investigating this further and in an attempt to ascertain whether there was a threshold value below which exacerbation did not occur, experiments were carried out with reoxygenation at a series of different oxygen tensions. Following reoxygenation, the total number of units of enzyme activity released to the perfusate over a 30 min period was determined and was related to the oxygen content of the perfusate. The results (Figure 8) reveal that as the oxygen tension increases so does the extent of exacerbation. For example, in the absence of reoxygenation (POs less than 5 mm Hg), 13000 mIU of HBDH are released in 30 min, reoxygenation with 10% 02 (PO2 approximately 70 mm Hg) increased the release to 19000 mIU/30 min; 20% O2 (POa approximately 140 mm Hg) gave 26000 mIU/30 min; 40% 0s (POs approximately 280 mm Hg) gave 36000 mIU/30 min; 60% 0s (PO2 approximately 430 mm Hg) gave 44000 mIU/30 min; 70% 0s (PO2 approximately 500 mm Hg) gave 57000 mIU/30 min; 80% 0s (PO2 approximately 570 mm Hg) gave 66000 mIU/SO min and 95% 02 (PO2 approximately 680 mm Hg) gave 112000 mIU/30 min. In all experiments at the end of the 30 min period of reoxygenation, the hearts were further reoxygenated with 95% 02. In the case of the OS, lo%, 20x, 40% and 45% 02 this led to an additional exacerbation of release to give a total release similar to that which would have been obtained with a single reoxygenation with
478
D. J. HEARSE
120 000
AND
S. M.
HUMPHREY
l-
I I O 0
I 200 PO,
mmHg
I
I
400
600
foweoxygenafion
FIGURE 8. The exacerbation of HBDH release from the isolated perfused anoxic (150 min) rat heart after reoxygenation at different oxygen tensions. Hearts were reoxygenated for 30 min and the total enzyme released (mIU) to the perfusion fluid was determined. For details see text. Each point represents the mean for four hearts and the bars represent the S.E.M.
higher oxygen concentrations. It therefore appears that the extent of exacerbation of enzyme release induced by reoxygenation is dependent upon the concentration of 0s and furthermore it cannot be overcome by a process of gradual reoxygenation.
4. Discussion The possibilities and limitations of metabolic protection of the ischemic myocardium have been discussed for many years. From the early clinical studies of Sodi-Pallares [27] into the use of glucose, potassium and insulin there have been many clinical and experimental studies into possible methods by which the myocardium could be supported during ischemia or anoxia. Many of these studies [8, 12, 22, 301 have further underlined the potential value of metabolic protection. The studies reported in this paper have utilized the anoxic and hypoxic isolated perfused rat heart. It is clearly appreciated [5] that this model differs from the ischemic heart segment in many respects, despite the inadequacies of the model it is
METABOLIC
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noteworthy that the characteristic enzyme release profiles observed in these and other studies [I, 61 have features in common with the profiles from ischemic dog and human hearts. For example, creatine phosphokinase activity is detected first and appears at a faster rate and in larger amounts than the other enzymes. a-Hydroxybutyrate dehydrogenase appears later but the release is maintained for a longer period of time. Using enzyme release as an index of myocardial damage the results described in this paper illustrate that with suitable interventions it is possible to considerably modify the pattern of cardiac enzyme release. It is however, important to question whether any reduction in enzyme release represents a true metabolic protection with improved viability or whether enzyme release has only been delayed or prevented, possibly by some physical effect, without enhancing the survival of the cell. The results show that the inclusion of glucose in the perfusion fluid during anoxia increases the duration of anoxia that can be tolerated without enzyme release and also reduces the rate of enzyme release once initiated. Although the inclusion of glucose at the time of reoxygenation does not greatly reduce the exacerbation of release, if glucose is included in the anoxic perfusion fluid for the entire anoxic period the reoxygenation phenomenon is prevented. These results would suggest that glucose exerts a true protective effect. We would suggest that this is a metabolic effect brought about by the ability of glucose to act as an energy source during anoxia. Under similar conditions we have shown [5, 61 that the glucose increases the myocardia1 level of ATP and also the ability of the heart to survive during, and recover from, anoxia. The increased availability of ATP would facilitate an extended period of cellular maintenance and prevent the deterioration which is intensified by reoxygenation. The possibility cannot be excluded that the protective effect of glucose is not entirely metabolic in origin and there may be some contribution from osmotic effects [18]. The results with graded hypoxia were very similar to those with glucose. An increasing availability of oxygen afforded increasing degrees of protection. This protective effect was sufficient to prevent the reoxygenation phenomenon. As with glucose, the limited availability of oxygen would facilitate increased ATP production, this time through a partially operative oxidative metabolism. The effects of glucose and hypoxia were shown to be additive and further increased the protective effect. With methyl prednisolone no clear protective effect was observed, while there was some reduction of enzyme release this was variable and the inclusion of methyl prednisolone during anoxia was not able to prevent the reoxygenation phenomenon. These results coupled with the inability of the compound to increase the functional recovery of the working heart after 30 min anoxia would suggest that methyl prednisolone is unable to improve cell viability and therefore does not offer a true protective effect. However, Libby et al. [19] have reported that hydrocortisone administration, in addition to reducing CPK loss in the infarcting dog heart,
480
D.J.HEARSE
AND S.M.HUMPHREY
also reduces the ST-elevation and the ultrastructural damage associated with myocardial ischemia. The reoxygenation phenomenon appears to represent the compression of damage into a few minutes which would normally have taken several hours. The effect cannot be prevented by the inclusion of protective agents at the time of reoxygenation, the inclusion of antioxidants or the use of gradual reoxygenation. Once the cell has deteriorated beyond a certain point enzyme release cannot be reduced and the reoxygenation phenomenon cannot be prevented. The action of protective agents is to prevent the cell deteriorating to the point where it becomes susceptible to damage. We would suggest that protective agents introduced during the initial phase of the reversible damage [II] will be effective in reducing cellular damage and thus delay the onset of irreversible damage. However once irreversible damage is initiated little can be done to prevent tissue deterioration and the cells become susceptible to effects such as the reoxygenation phenomenon. The absence of a major increase in enzyme release following reoxygenation during phase 1 release [7, 81 may indicate that irreversible damage to the bulk of the myocardium has not occurred and that the occurrence of irreversible damage is represented by the onset of phase 2 release. These results would underline the importance of the need for the rapid introduction of protective agents to the anoxic or hypoxic myocardium. The sooner the introduction after the onset of hypoxia the greater will be the chance of significant metabolic protection. Protection must be initiated before the transition from reversible to irreversible cellular damage and this may perhaps be best achieved by interventions aimed at reducing cellular energy demands and maximizing cellular energy production.
Acknowledgements
This work was carried out with the aid of a grant from the British Heart Foundation. We acknowledge the assistance of Caroline Tahourdin in the methyl prednisolone recovery studies and David Stewart for computer programming.
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