Journal of Molecular and Cellular Cardiology (1975) 7, 827-840
Enzyme
K. SAKAI,
Release Resulting in the Isolated, M. M. GEBHARD,
from Total Ischemia and Reperfusion Perfused Guinea Pig Heart
P. G. SPIECKERMANN
AND H. J. BRETSCHNEIDER
Department of Physiology, University of Giittingen, 34 Giittingen, West Germany (Received 25
June 1974, accepted in revisedform
14 January
1975)
K. SAIGU, M. M. GEBHARD, P. G. SPIECKERMANN AND H. J. BRETSCHNEIDER. Enzyme Release Rusulting from Total Ischemia and Reperfusion in the Isolated, Perfused Guinea Pig Heart. 3oumd of Molecular and Cellular Cardiology (1975) 7,827-840. A study of enzyme release induced by total myocardial ischemia was undertaken using isolated guinea pig hearts perfused at constant flow (4 ml/ min) . Mechanical activity (perfusion pressure and heart rate) and per&ate enzyme activity (malate dehydrogenase, lactate dehydrogenase and creatine phosphokinase) were examined throughout experiments in which the duration of total ischemia was varied (5, 10 and 20 min). The enzyme releases occurred generally in a biphasic pattern; a marked release just after reperfusion (first phase) and later a moderate release (second phase). The level of enzyme activity returned close to the control level within 7 min. The first phase of release was not accompanied by resumption of contraction. The rate of enzyme release correlated closely with the duration of total ischemia. The present results suggest that enzyme release during/after the tested durations of ischemia may not necessarily reflect serious myocardial cell damage, but rather alterations of the membrane permeability of myocardial cells. KEY WORDS: Isolated Malate dehydrogenase; permeability.
perfused Lactate
guinea pig heart; Total dehydrogenase; Creatine
ischemia; Enzyme release; phosphokinase; Membrane
1. Introduction Recent developments in diagnostic enzymology have established the concept that pathological conditions including ischemia related to myocardial infarction cause abnormal elevations of serum enzyme activity [II, 221. Karmen et al. [9] reported elevated serum levels of glutamate oxaloacetate transaminase following myocardial infarction. Similar increases were also observed in other enzymes such as malate dehydrogenase [21], lactate dehydrogenase [25] and creatine phosphokinase [4]. Thus, the elevation of several serum enzymes has become an important criterion in the diagnosis of acute myocardial infarction. Although the increased release of enzymes has been considered to be due to a change in membrane permeability with respect to enzymes and/or irreversible alteration of cells, the mechanism is not immediately evident. The present study using isolated perfused hearts is concerned with determination of experimental conditions and enzyme release following total ischemia. It also seemed of special
828
K. SAKAI ET-AL.
interest to deduce whether enzyme release under ments is due to reversible or irreversible myocardial
2. Materials
the present ischemia cell damage.
experi-
and Methods
One hundred and seven adult guinea pigs of both sexes, weighing around 300 g and fed standard laboratory diet, were used. The animals were injected with heparin (5 mg) intraperitoneally 1 h before killing and stunned by a blow on the head. In 61 experiments, the hearts were excised and washed in ice-cold, Oa-saturated Tyrode solution (pH 7.4)) immediately after thoracotomy. After a cannula filled with the solution was inserted into the aorta, the heart was mounted in a water-jacketed chamber and perfused according to the Langendorff technique at constant flow (4 ml/ min) by means of a peristaltic pump (Desaga, Heidelberg, West Germany). For the remaining46experiments, theaortawas exposedafter thoracotomyand clampedwith the transient a bulldog arterial clip about 5 mm from the ventricle. To minimize myocardial ischemia during the operation, 5 ml of an ice-cold cardioplegic solution [,!?I were injected over a period of 1 min through the aorta into the coronary system with a syringe. The solution contained the following substances (in mM/l) : NaCl, 12; NaOH, 1; MgCla, 0.8; KCl, 7.3; procaine hydrochloride, 7; and D-mannit, 270; (a total concentration of about 320 mOs/l), and was saturated thoroughly with pure oxygen. The pH was maintained at 7.4. Following the injection of the solution, contraction stopped within a few seconds. The excised heart was rinsed in the ice-cold, OS-saturated cardioplegic solution. After a cannula filled with the solution was inserted into the aorta, the heart was mounted in the perfusion system and perfused with different media (Table 1). The glucose concentration of the perfusates was 6 mM unless otherwise noted. These perfusates were aerated thoroughly with 95% Ca:5o/o COa. The temperature of the perfusion medium was 35°C as it entered the heart. Unless the heart had been damaged during the operation, it began to beat strongly within 90 s after starting the perfusion. This delay may be related to the use of cardioplegic solution and/or the temperature effects. The pH of the perfusate was routinely checked with a pH Meter E 300 B (Metrohm, Herisau, Switzerland). Perfusate oxygen content was determined by analyzing samples with a gas analyser, Lex-Oa-Con. (Lexington Inst. Corp., Waltham, Massachusetts, USA). The osmolarity and electrolytes were determined by using a micro-osmometer (Knauer, Berlin, West Germany) and an atomic absorption spectrophotometer (Perkin-Elmer 403, Pomona, California, USA), respectively. Perfusion pressure was measured with Statham pressure transducer (Statham Inst. Inc., Oxnard, California, USA) and recorded continuously (Hellige, Mr. 76, West Germany). Heart rate was routinely determined with a stop watch. The enzymes in the effluent perfusate were assayed kinetically at 25°C with a filter photometer (366 nm) (Eppendorf, West Germany). Malate dehydrogenase (MDH) activity was determined by the method of Bergmeyer and Bernt [I].
ENZYME
RELEASE
AND
MYOCARDIAL
ISCHEMIA
829
Creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) activities were determined by the methods of Oliver [14] and Wroblewski and Ladue [25], respectively. Lactate was measured by the method of Gutmann and Sahlefeld [S]. The agents used were supplied by Bijhringer Mannheim (West Germany). Enzyme amount released into the perfusates was expressed as follows: mU/(min x g wet weight) = mu/ml x 4 ml/min x l/(5 x g dry heart weight after perfusion). Six non-perfused hearts were used for the calculation of control values of dry and wet heart weights. The dry weight was obtained as 20.86 f 0.190/, (s.E.M.) (n = 6) of the wet weight. The wet weight prior to perfusion was calculated by multiplying the dry weight after perfusion with the factor 5. All results were expressed as means 5 standard error of the mean (S.E.M.j (number of experiments). The statistical significance of differences between means was calculated with the Wilcoxon, Mann and Whitney test (U-test) or the Kruskal and Wallis test (H-test) [28]. 3. Results Selection of coronary flow rate Hearts not treated with cardioplegic solution were perfused at different mean coronary flow rates (4, 6 and 8 ml/ min) with Tyrode solution, and the effects on lactate production, MDH release and mechanical activity (perfusion pressure and heart rate) were studied. One hour after perfusion at these rates, the values of perfusion pressure and heart rate from each group of four hearts (n = 4) were as follows: 26 f 3 mmHg and 162 & 2 min-1 at 4 ml/min; 28 & 2 mmHg and 164 f 3 min-1 at 6 ml/min; and 30 + 1 mmHg and 160 & 2 min-1 at 8 ml/min. These values are not significantly different (P > 0.05), although perfusion pressure tended to rise with flow rate increase. Table 2 shows the effects of variations in the flow rate on lactate output and MDH release. Just after perfusion at the tested rates, relatively high lactate output and MDH release were observed. Stable levels were obtained after 10 min of perfusion. At different time intervals following the perfusion at the tested flow rates, there were no significant differences in either the rates of lactate output or the MDH release. Because myocardial oxygen consumption increases as perfusion pressure rises [12], we selected the flow rate of 4 ml/mm for the stability of preparations. Myocardial enzyme release and mechanical activity (p erf usion pressure and heart rate) for hearts treated with cardioplegic solution and hearts not treated The enzyme release and mechanical activity were compared between the hearts treated with cardioplegic solution and those that were not (Figure 1). The nontreated hearts stopped beating at 16 f 2 s (n = 24) after immersion in ice-cold
830
K. SAKAI
TABLE
1. Perfusate composition
Composition
Tyrode
Na+ K+ Ca*+ Mgs’ ClHCOsHzPOr
Krebs-Henseleit (n-w
150.0 2.7 1.9 1.0 141.6 11.9 0.5
s04a-
4.0 6.0 12.0 320
Glucose Osmolarity WW)
TABLE
El-AL.
Locke
143.0 5.9 2.5 1.2 125.2 25.0 1.2 1.2
155.9 5.6 1.6 161.2 1.8 -
6.0
6.0
315
332
2. Effects of different flow rates on lactate output and MDH release Time after perfusion (min)
Flow rate (ml/min) 4 4 6 8
0.53 f 0.11 0.37 f 0.06 0.38 & 0.08
P
N.S.
3 4 6 8
52.95 f 11.78 37.34 & 7.66 54.57 -+ 16.36
P
N.S.
10 30 Lactate formed (pmol/min . g) 0.25 f 0.03 0.29 f 0.08 0.21 f 0.04 0.25 f 0.06 0.22 f 0.04 0.31 5 0.05 N.S.
N.S.
9 29 MHD released (mU/min . g) 28.99 f 5.71 27.62 & 5.98 21.33 h 6.06 21.33 & 6.06 26.57 & 1.31 26.57 & 1.31 N.S.
N.S.
60 0.20 &- 0.03 0.22 f 0.05 0.23 f 0.01 N.S.
59 22.47 f 20.23 & 24.57 -f
2.90 4.90 1.31
N.S.
Three groups each of four hearts not treated for cardioplegia were studied at each different flow rate. Following perfusion aliquotes were collected for 1 min at each time interval. The amounts of lactate and MDH were expressed in terms of gram wet heart weight before perfusion (see methods). The values given are means & S.E.M. The H-test was used to evaluate the significance of the differences between each of the groups perfused at the different flow rates. N.S. not significant (P > 0.05). The means of body and dry weights of hearts from 12 animals were 320.0 f 8.9 g and 0.21 f 0.02 g, respectively.
ENZYME
RELEASE
AND
MYOCARDIAL
g 100I-
831
ISCHEMIA
LDH
CPK
r-+e--e-e _
-By 0 0
30
I 60
I 90
120
150
_ 180
-210 240
270
Q 308
(min)
FIGURE 1. Enzyme release and mechanical activity in the hearts treated or not treated for cardioplegia. Changes in enzyme releases (MDH, LDH and CPK), perfusion pressure (PP) and heart rate (HR) were examined over 5 h. Vertical bars indicate means f S.E.M. (0) Cardioplegia (n = 13); (0) Non-treated (n = 6).
Tyrode solution. Following perfusion with warm Tyrode solution, contractions began at 23 f 2 s. The initial enzyme release following perfusion stabilized within 30 min. Heart rate remained constant throughout the study, but perfusion pressure rose progressively. The treated hearts usually stopped beating in the diastole at 5.6 & 0.3 s (rr = 46) following the injection of cardioplegic solution. Strong contractions began at 54 f 4 s following exposure to warm Tyrode solution. Stable levels of enzyme release and mechanical activity were reached within 30 to 60 min after the beginning of perfusion, and were maintained for at least 5 h. Concerning each enzyme release there were no systematic differences between the groups treated and not treated for cardioplegia. However, higher levels in heart rate and perfusion pressure were observed in the non-treated group. Perfusate selection
The treated hearts were perfused with Krebs-Henseleit, Locke and Tyrode solutions, respectively (Table 1). Figure 2 shows the enzyme activities in the effluent fluid and mechanical activities.
832
K. SAKAI
500
-T
ET AL.
MDH
400 300 200 100 7 i 2 E
0 200
LDH
-T
100 0 400 300 200 100 0
Tc 5
HR
150 100 50
PP 2
60
+-.--o-o-o-(
1
30 0
@q+-^e~: I I 30 60
0
I 90
I 120
I 150
I I8
(min)
FIGURE 2. Enzyme release and mechanical activity in the heart preparations perfused with Krebs-Henseleit, Locke and Tyrode solutions, respectively. The hearts were subjected to cardioplegic treatment. Changes in enzyme releases (MDH, LDH and CPK), perfusion pressure (PP) and heart rate (HR) were examined over 3 h. Vertical bars indicate means 6 S.E.M. (0-O) Locke (R = 4); (H) Krebs-Henseleit (n = 9); (@- - - -0) Tyrode (n = 13).
Even after 30 min of perfusion using Krebs-Henseleit solution considerable enzyme release was observed, with the exception of LDH. Although a steady state level of MDH and CPK release was obtained about 90 min after the start of perfusion, the enzyme levels tended to rise again. Perfusion pressure and heart rate also rose gradually. Perfusion with Locke solution induced more pronounced enzyme release than with Krebs-Henseleit solution. Ninety minutes after starting the perfusion a steady state
ENZYME
RELEASE
AND
MYOCARDIAL
833
ISCHEML4
of enzyme release was obtained at relatively high level. Progressive rise in perfusion pressure and heart rate was also observed. It was difficult to maintain the pH of 7.4 constantly during the perfusion. A steady level of enzyme release was reached later with Krebs-Henseleit or Locke solution than with Tyyrode solution. It might be finally concluded that a stable level of both enzyme release and mechanical activity was more rapidly arrived at with Tyrode perfusion, and it was therefore more suitable for the present study.
400,
300 c%s
I MDH
I
LDH --o
HR
50 100
50 0
PP 0
30
60
SO 120 150 160 210 240 270 300
FIGURE 3. Effects of variations inthe glucose concentration of Tyrode solution on enzyme release and mechanical activity. Glucose was added in the solution at 4, 6 and 12 mre concentrations. Changes in enzyme release (MDH, LDH and CPK), perfusion pressure (PP) and heart rate (HR) were examined over 5 h. Vertical bars indicate means f S.E.M. (ha) 4 mr+ Glucose (a = 4) ; (@- - +) 6 m~/l Glucose (a = 13); (O-0) 12 mM/l Glucose (a = 4).
Tyrode solution was modified by the addition of different concentrations of glucose. Figure 3 summarizes the effect of alterations in the glucose concentrations (4, 6 and 12 mM) of perfusate on enzyme release and mechanical activity. After the beginning of perfusion, the perfusate containing 12 mM glucose induced more marked enzyme release (especially MDH and CPK) than those with 4 and 6 mM glucose, and did not rapidly produce a steady level. Even after 1 h of perfusion, the values for MDH and CPK release at 12 mM glucose were significantly higher than those at 4 and 6 mM glucose (P < 0.001). Although changes in enzyme
834
K. SAKAI
ETAL.
release after perfusion with 4 and 6 mM glucose followed a similar pattern, a more stable level seemed to be induced by that with 6 mM glucose. In all cases, perfusion pressure and heart rate remained at a constant level throughout the study.
Pre-ischemia
(60
Pump stop
Reperfusion
i
i Post-ischemia
lschemia
min )
w’)I min u-1
I t t Control (Sampling)
t Onset of perfusion
’
I
I’
I
I
I
I
I
I
2’
3’
4’
5’
6’
7’
L
Sampling
t
FIGURE 4. Diagram of perfusion plan under the ischemic experiments.
Enzyme releasefollowing total ischemia The effect of cardioplegia on enzyme release was tested in experiments with 20 min ischemia. After a 60 min equilibration period hearts were subjected to total ischemia by stopping the perfusion pump (Figure 4). Following reperfusion aliquotes were TABLE
3. Appearance reperfusion
Duration of &hernia Incidence of arrest Per cent Begin of recontraction
of cardiac arrest following
(min)
(s)
ischemia and recontraction
following
5
10
20
4113 31
516 83 39.0 5 2.9*
616
30.7 f 1.6*
100
46.7 + 5.6
Hearts not treated for cardioplegia were subjected to total ischemia of different periods. Mean time interval for the appearance of cardiac arrest following initiation of ischemia; 6.9 & 2.6 min (n = 15). The values marked with asterisk were calculated only for the preparations, in which cardiac arrest occurred after ischemia. Values represent means 5 S.E.M. These values differ significantly with P < 0.001 (H-test). continuously collected and sampled at the following intervals: 30”, 1’) 2’, 3’, 4’, 5’, 6’, 7’. The sample containers were replaced by clean, fresh tubes at the termination of each interval. The activities represent the integral enzyme releases for the successive intervals. As observed in Figure 5 and Table 4, the hearts treated with cardioplegic solution showed significantly less enzyme release compared with the non-treated. The cardioplegic solution was not used in a series of ischemic studies, because it possessed some influence on enzyme release even after 1 h of perfusion.
ENZYME
RELEASE
AND
MYOCARDIAL
MDH
0 Cardioplegia 0 Non-treated
0
I
2
3 (min)
4
835
ISCHEMIA
5
(n=6) (n:6)
6
7
FIGURE 5. Myocardial enzyme release after 20 min of total &hernia. After 60 mm-equilibration period total ischemia was induced by stopping perfusion pump. Vertical bars indicate means & S.&M. for enzyme amount released following reperfusion. Each value was expressed in per cent increase over pm-ischemia control. The maximum values (first phase) of individual enzymes (MDH, LDH and CPK) were significantly different from the corresponding control values (P < 0.001). Two minutes after reperfusion (second phase), only each value of LDH and CPK in the hearts not treated for cardioplegia was significantly different from the corresponding control value (P < 0.1) ; (0) Cardioplegia (a = 6); (0) Non-treated (n = 6).
Table 4 indicates the results of enzyme release for various durations of total ischemia: 5, 10 and 20 min. The releases generally occurred in a biphasic pattern: a marked release immediately after reperfusion (first phase) and later a moderate release (second phase) (Figure 5). In the first phase the maximum release of MDH, LDH and CPK occurred within 30 s following reperfusion. During this time the resumption of contraction was not yet observed (Table 3). The releases were transient and the level returned close to the control levels within 60 s; after that a
836 TABLE
K. SAUAI ETAL.
4. Myocardial
enzyme release after different ischemic durations Cardioplegia
No cardioplegia Duration of &hernia (min) ~--
5 (n = 13)
20 (n = 6)
10 (n = 6)
MDH LDH CPK
225.7 f 18.1 240.0 & 23.4 274.7 f 30.0
Maximum 581.4 5 115.1 449.2 & 33.2 628.5 f 72.7 429.0 + 50.4 711.8 5 117.6 438.8 & 51.7
MDH LDH CPK
299.0 f 34.3 350.7 f 50.4 313.2 f 53.3
584.7 & 30.1 560.8 f 54.6 481.0 f 99.1
20 (n = 6) 310.6 + 75.6 150.8 & 18.7 233.2 & 10.0
Total 798.8 & 72.3 1195.8 f 195.0 1035.8 f 310.9
411.2 + 93.0* 279.0 f 56.4** 322.2 f 18.4**
Each value was expressed in per cent increase over the control value. Maximum : maximum enzyme release in the first phase. Total: total enzyme release into the effluent perfusate from the hearts during 5 min after reperfusion. Each value is mean & S.E.M. Values marked with asterisks in the table were significantly different (u-test) from those of the corresponding 20 min ischemia group not treated for cardioplegia.* P < 0.025;** :P < 0.005. Total values in the group not treated for cardioplegia differ significantly from the other values in this group with P < 0.01 (MDH), P < 0.01 (LDH) and P < 0.05 (CPK), respectively (H-test). moderate release (second phase), always accompanied by the resumption of contraction, lasted for a few minutes. Even if the hearts were subjected to 20 min of total ischemia, lactate output and enzyme release as well as mechanical activity returned close to the control levels about 7 min after reperfusion (Table 5). The rate of each enzyme release and the time contraction began for all ischemic periods tested was dependent on the duration of ischemia, as shown in Tables 3 and 4. TABLE
5. Myocardial
metabolic changes before/after 20 min ischemia Time after reperfusion (min)
7 Lactate formed (pmol/min 0.20 & 0.03 0.26 f 0.06 Control
6 MDH released (mU/min 32.4 f 3.4 48.2 -& 7.8
15 / g) 0.20 f 0.03 14
Control
/ g) 39.3 & 6.4
Following reperfusion aliquots were collected for 1 min at each time interval. The amounts of lactate and MDH were expressed in terms of gram wet heart weight before perfusion (see methods). The values in this table represent means & S.E.M. for, respectively, four hearts not treated for cardioplegia. These values were not significantly different from the control means (P > 0.05) (U-test). Each control value was obtained within 2 min prior to ischemia. Perfusion pressure: control, 20.8 f 1.3 mmHg; min after reperfusion, 21.5 5 1.5 mmHg (P > 0.05). Heart rate: control, 154.0 f 4.8 min- 1; 7 min after reperfusion, 158.5 f 7.9 min-l (P > 0.05).
ENZYME
RELEASE
AND
MYOCARDIAL
ISCHEMIA
837
4. Discussion The present results show that brief, total myocardial ischemia induces pronounced but transient enzyme release from the myocardium. The release need not necessarily be related to irreversible cell damage. Prior to the ischemic studies it was important to establish the most suitable experimental conditions. Shipp et al. [20], using isolated working rat heart, have reported that perfusion rates of 5 ml/min or higher are needed to avoid metabolic changes suggestive of oxygen deficiency. The present study, however, showed that the different coronary flow rates ranging from 4 to 8 ml/min induced no significant differences on the rates of lactate output. These values were in accordance with those calculated from another study [1.5] at a comparable flow rate. According to Fisher and Williamson using rats of 200 to 260 g [5], there was an adequate oxygen supply at 4 ml/min perfusion rate with oxygen saturated solution and further elevation of oxygen extraction with 2,4-dinitrophenol. It was tried to minimize the myocardial energy demand during the operation, not only by decreasing myocardial temperature but also by using the cardioplegic solution [2]. The myocardial oxygen deficiency, which occurs during the operation, might induce the release of enzymes. The myocardial oxygen demand is reduced from the normal heart condition by a factor of about 100 when the cardioplegic solution is used at 5°C. In fact, a more stable condition in the preparations treated with the cardioplegic solution was maintained over a longer period in comparison with the non-treated. Effects of several perfusates (Tyrode, Krebs-Henseleit and Locke solution) were compared in the hearts treated with the cardioplegic solution. Tyrode solution had the most suitable effect on enzyme release as well as on mechanical activity, which has been shown to be an indication of myocardial energy demand [12]. Tyrode solution maintained a stable state of the preparation for at least 5 h (Figure 1). Several workers have reported protective effects of glucose on ischemic and/or anoxic hearts [lo, 161. Therefore, the modification in the concentration of glucose in the perfusate might be interesting. As observed, a more stable level seemed to be produced by the perfusion with 6 mM glucose (Figure 3). Under 20 min ischemia, the induced enzyme releases for the group treated for cardioplegia were smaller than for the non-treated group. This finding can be explained by the reduction of myocardial oxygen demand due to cardioplegia and/or a membrane stabilizing effect of procaine. Under clinical conditions, procaine in a cardioplegic solution will be rapidly inactivated by blood esterases [17], whereas in the present study it seems to decrease membrane permeability to myocardial enzymes by close binding with the cell membrane over a long period. Under different durations of total ischemia, enzyme release generally occurred in a biphasic pattern; a marked release just after reperfusion (first phase) and later a moderate release (second phase). The first phase of release occurred without the
838
K. SAKAI
ETAL.
concomitant resumption of contraction. De Leiris et al. [3] have stated that cardiac contraction is a major determinant of enzyme release into the perfusion fluid. However, under the present ischemic conditions, the resumption of contractility was not necessarily needed for enzyme release. In clinical practice it is believed that elevations of serum enzymes following myocardial &hernia are related quantitatively to enzyme release from irreversibly injured cells [22]. However, the mechanism of enzyme release is still controversial. Hearse et al. [7] have stated that cellular leakage of enzymes occurs when the cell structure is damaged. It has also been quoted by De Leiris et al. [3] that enzyme release entails an increase of membrane permeability. Zierler [27] also suggested that enzymes could be released through an alteration of membrane permeability under some conditions. According to our present results, the increased enzyme release appearing after total ischemia for the tested durations might not necessarily result from irreversible cell damage, but rather from a transient increase in membrane permeability to myocardial enzymes during/after ischemia. The relatively rapid recovery of mechanical activity as well as enzyme release and lactate output after 20 min of total ischemia definitely suggests this possibility, although Hearse and Stewart [8], using isolated working rat hearts subjected to a comparable ischemic period, deduced opposite conclusions. Despite some differences in methods and species, our present views would be supported by the conclusions of Neely et al. [13] and others [23] in which permanent myocardial damage did not occur if the duration of ischemia was less than 20 min. Experimental and clinical findings would apparently seem to lend further support to our views. It is known that changes in coronary arterio-venous differences in enzymes occur within 20 to 30 min after the onset of local oxygen deficiency in the myocardium [19, 261. As previously reported, there is a close correlation between the rate of enzyme release and myocardial high energy phosphate content [24] ; appreciable amounts of enzyme proteins released from myocardial cells into the coronary effluent were observed after only a small decrease of myocardial ATP. At this stage myocardial cells did not yet sustain irreversible damage. The more the myocardial ATP content decreased, the more the rate of enzyme release increased. The results presented in Table 4 might partly be accounted for by the above description. It remains to be studied whether there is a direct causal relationship between changes in energy metabolism and the liberation of enzymes. Acknowledgments We are very grateful to Mrs R. Hahn, Mrs G. Sander and Miss A. Holtmeyer for their excellent assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 89, Kardiologie Gottingen). A part of this study will be presented as an inaugural dissertation of the University of Gottingen by M. M. Gebhard.
ENZYME RELEASE AND MYOCARDIAL
ISCHEMIA
839
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ETAL.
SHIPP, J. C., MATOS, 0. E., KNIZLEY, H. & CREVASSE, L. E. COz formed from endogenous and exogenous substrates in perfused rat heart. Americun 30u7nal c$ Physiology 207, 1231-1236 (1964). SIEGEL, A. & BING, R. J. Plasma enzyme activity in myocardial infarction in dog and man. Proceedings of the Society for Experimental Biology and Medicine 91, 604-607 (1956). SOBEL, B. E. & SHELL, W. E. Serum enzyme determinations in the diagnosis and assessment of myocardial infarction. Circulation 45, 47 l-482 (1972). SPIECKERMANN, P. G. uberlebens- und Wiederbelebungszeit des Herrens. (Anaesthesiology and Resuscitation, Vol. 66). pp. 5-6. Berlin, Heidelberg, New York: Springer Verlag (1973). SPIECKERMANN, P. G., GEBHARD, M. M., KALBOW, K., KNOLL, D., KOHL, F., NORDBECK, H., SAKAI, K. & BRFTXHNEIDER, H. J. Freisetzung von Enzymen aus der Herzmuskelzelle wihrend Sauerstoffmangel. Verhandlungen der Deutschen Gesellschaft f$r Kreislaufforschung 39, 193-198 (1973). WROBLEWSKI, F. & LADUE, J. S. Lactic dehydrogenase activity in blood. Proceedings of the Society for Experimental Biology and Medicine 90, 2 IO-2 13 (1955). ZASVORKA, F. Z., HOFFMANN, E., RUMPF, K. D., WALTER, P., GRGGLER, F., BEDDERMANN, C., LAMPRECHT, W. & BORST, H. G. Aenderungen der Serum LDH- und GOTIsoenzyme wshrend temporlrer MyokardischBmie. In Vascular Smooth Muscle. E. Betz Ed. pp. 168-170. Berlin, Heidelberg, New York: Springer Verlag (1972). ZIERLER, K. L. Muscle membrane as a dynamic structure and its permeability to aldolase. Annals New 2’ork Academy of Sciences75, 227-234 (1958/59).
Enzyme
K. SAKAI,
Release Resulting in the Isolated, M. M. GEBHARD,
from Total Ischemia and Reperfusion Perfused Guinea Pig Heart
P. G. SPIECKERMANN
AND H. J. BRETSCHNEIDER
Department of Physiology, University of Giittingen, 34 Giittingen, West Germany (Received 25
June 1974, accepted in revisedform
14 January
1975)
K. SAIGU, M. M. GEBHARD, P. G. SPIECKERMANN AND H. J. BRETSCHNEIDER. Enzyme Release Rusulting from Total Ischemia and Reperfusion in the Isolated, Perfused Guinea Pig Heart. 3oumd of Molecular and Cellular Cardiology (1975) 7,827-840. A study of enzyme release induced by total myocardial ischemia was undertaken using isolated guinea pig hearts perfused at constant flow (4 ml/ min) . Mechanical activity (perfusion pressure and heart rate) and per&ate enzyme activity (malate dehydrogenase, lactate dehydrogenase and creatine phosphokinase) were examined throughout experiments in which the duration of total ischemia was varied (5, 10 and 20 min). The enzyme releases occurred generally in a biphasic pattern; a marked release just after reperfusion (first phase) and later a moderate release (second phase). The level of enzyme activity returned close to the control level within 7 min. The first phase of release was not accompanied by resumption of contraction. The rate of enzyme release correlated closely with the duration of total ischemia. The present results suggest that enzyme release during/after the tested durations of ischemia may not necessarily reflect serious myocardial cell damage, but rather alterations of the membrane permeability of myocardial cells. KEY WORDS: Isolated Malate dehydrogenase; permeability.
perfused Lactate
guinea pig heart; Total dehydrogenase; Creatine
ischemia; Enzyme release; phosphokinase; Membrane
1. Introduction Recent developments in diagnostic enzymology have established the concept that pathological conditions including ischemia related to myocardial infarction cause abnormal elevations of serum enzyme activity [II, 221. Karmen et al. [9] reported elevated serum levels of glutamate oxaloacetate transaminase following myocardial infarction. Similar increases were also observed in other enzymes such as malate dehydrogenase [21], lactate dehydrogenase [25] and creatine phosphokinase [4]. Thus, the elevation of several serum enzymes has become an important criterion in the diagnosis of acute myocardial infarction. Although the increased release of enzymes has been considered to be due to a change in membrane permeability with respect to enzymes and/or irreversible alteration of cells, the mechanism is not immediately evident. The present study using isolated perfused hearts is concerned with determination of experimental conditions and enzyme release following total ischemia. It also seemed of special
828
K. SAKAI ET-AL.
interest to deduce whether enzyme release under ments is due to reversible or irreversible myocardial
2. Materials
the present ischemia cell damage.
experi-
and Methods
One hundred and seven adult guinea pigs of both sexes, weighing around 300 g and fed standard laboratory diet, were used. The animals were injected with heparin (5 mg) intraperitoneally 1 h before killing and stunned by a blow on the head. In 61 experiments, the hearts were excised and washed in ice-cold, Oa-saturated Tyrode solution (pH 7.4)) immediately after thoracotomy. After a cannula filled with the solution was inserted into the aorta, the heart was mounted in a water-jacketed chamber and perfused according to the Langendorff technique at constant flow (4 ml/ min) by means of a peristaltic pump (Desaga, Heidelberg, West Germany). For the remaining46experiments, theaortawas exposedafter thoracotomyand clampedwith the transient a bulldog arterial clip about 5 mm from the ventricle. To minimize myocardial ischemia during the operation, 5 ml of an ice-cold cardioplegic solution [,!?I were injected over a period of 1 min through the aorta into the coronary system with a syringe. The solution contained the following substances (in mM/l) : NaCl, 12; NaOH, 1; MgCla, 0.8; KCl, 7.3; procaine hydrochloride, 7; and D-mannit, 270; (a total concentration of about 320 mOs/l), and was saturated thoroughly with pure oxygen. The pH was maintained at 7.4. Following the injection of the solution, contraction stopped within a few seconds. The excised heart was rinsed in the ice-cold, OS-saturated cardioplegic solution. After a cannula filled with the solution was inserted into the aorta, the heart was mounted in the perfusion system and perfused with different media (Table 1). The glucose concentration of the perfusates was 6 mM unless otherwise noted. These perfusates were aerated thoroughly with 95% Ca:5o/o COa. The temperature of the perfusion medium was 35°C as it entered the heart. Unless the heart had been damaged during the operation, it began to beat strongly within 90 s after starting the perfusion. This delay may be related to the use of cardioplegic solution and/or the temperature effects. The pH of the perfusate was routinely checked with a pH Meter E 300 B (Metrohm, Herisau, Switzerland). Perfusate oxygen content was determined by analyzing samples with a gas analyser, Lex-Oa-Con. (Lexington Inst. Corp., Waltham, Massachusetts, USA). The osmolarity and electrolytes were determined by using a micro-osmometer (Knauer, Berlin, West Germany) and an atomic absorption spectrophotometer (Perkin-Elmer 403, Pomona, California, USA), respectively. Perfusion pressure was measured with Statham pressure transducer (Statham Inst. Inc., Oxnard, California, USA) and recorded continuously (Hellige, Mr. 76, West Germany). Heart rate was routinely determined with a stop watch. The enzymes in the effluent perfusate were assayed kinetically at 25°C with a filter photometer (366 nm) (Eppendorf, West Germany). Malate dehydrogenase (MDH) activity was determined by the method of Bergmeyer and Bernt [I].
ENZYME
RELEASE
AND
MYOCARDIAL
ISCHEMIA
829
Creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) activities were determined by the methods of Oliver [14] and Wroblewski and Ladue [25], respectively. Lactate was measured by the method of Gutmann and Sahlefeld [S]. The agents used were supplied by Bijhringer Mannheim (West Germany). Enzyme amount released into the perfusates was expressed as follows: mU/(min x g wet weight) = mu/ml x 4 ml/min x l/(5 x g dry heart weight after perfusion). Six non-perfused hearts were used for the calculation of control values of dry and wet heart weights. The dry weight was obtained as 20.86 f 0.190/, (s.E.M.) (n = 6) of the wet weight. The wet weight prior to perfusion was calculated by multiplying the dry weight after perfusion with the factor 5. All results were expressed as means 5 standard error of the mean (S.E.M.j (number of experiments). The statistical significance of differences between means was calculated with the Wilcoxon, Mann and Whitney test (U-test) or the Kruskal and Wallis test (H-test) [28]. 3. Results Selection of coronary flow rate Hearts not treated with cardioplegic solution were perfused at different mean coronary flow rates (4, 6 and 8 ml/ min) with Tyrode solution, and the effects on lactate production, MDH release and mechanical activity (perfusion pressure and heart rate) were studied. One hour after perfusion at these rates, the values of perfusion pressure and heart rate from each group of four hearts (n = 4) were as follows: 26 f 3 mmHg and 162 & 2 min-1 at 4 ml/min; 28 & 2 mmHg and 164 f 3 min-1 at 6 ml/min; and 30 + 1 mmHg and 160 & 2 min-1 at 8 ml/min. These values are not significantly different (P > 0.05), although perfusion pressure tended to rise with flow rate increase. Table 2 shows the effects of variations in the flow rate on lactate output and MDH release. Just after perfusion at the tested rates, relatively high lactate output and MDH release were observed. Stable levels were obtained after 10 min of perfusion. At different time intervals following the perfusion at the tested flow rates, there were no significant differences in either the rates of lactate output or the MDH release. Because myocardial oxygen consumption increases as perfusion pressure rises [12], we selected the flow rate of 4 ml/mm for the stability of preparations. Myocardial enzyme release and mechanical activity (p erf usion pressure and heart rate) for hearts treated with cardioplegic solution and hearts not treated The enzyme release and mechanical activity were compared between the hearts treated with cardioplegic solution and those that were not (Figure 1). The nontreated hearts stopped beating at 16 f 2 s (n = 24) after immersion in ice-cold
830
K. SAKAI
TABLE
1. Perfusate composition
Composition
Tyrode
Na+ K+ Ca*+ Mgs’ ClHCOsHzPOr
Krebs-Henseleit (n-w
150.0 2.7 1.9 1.0 141.6 11.9 0.5
s04a-
4.0 6.0 12.0 320
Glucose Osmolarity WW)
TABLE
El-AL.
Locke
143.0 5.9 2.5 1.2 125.2 25.0 1.2 1.2
155.9 5.6 1.6 161.2 1.8 -
6.0
6.0
315
332
2. Effects of different flow rates on lactate output and MDH release Time after perfusion (min)
Flow rate (ml/min) 4 4 6 8
0.53 f 0.11 0.37 f 0.06 0.38 & 0.08
P
N.S.
3 4 6 8
52.95 f 11.78 37.34 & 7.66 54.57 -+ 16.36
P
N.S.
10 30 Lactate formed (pmol/min . g) 0.25 f 0.03 0.29 f 0.08 0.21 f 0.04 0.25 f 0.06 0.22 f 0.04 0.31 5 0.05 N.S.
N.S.
9 29 MHD released (mU/min . g) 28.99 f 5.71 27.62 & 5.98 21.33 h 6.06 21.33 & 6.06 26.57 & 1.31 26.57 & 1.31 N.S.
N.S.
60 0.20 &- 0.03 0.22 f 0.05 0.23 f 0.01 N.S.
59 22.47 f 20.23 & 24.57 -f
2.90 4.90 1.31
N.S.
Three groups each of four hearts not treated for cardioplegia were studied at each different flow rate. Following perfusion aliquotes were collected for 1 min at each time interval. The amounts of lactate and MDH were expressed in terms of gram wet heart weight before perfusion (see methods). The values given are means & S.E.M. The H-test was used to evaluate the significance of the differences between each of the groups perfused at the different flow rates. N.S. not significant (P > 0.05). The means of body and dry weights of hearts from 12 animals were 320.0 f 8.9 g and 0.21 f 0.02 g, respectively.
ENZYME
RELEASE
AND
MYOCARDIAL
g 100I-
831
ISCHEMIA
LDH
CPK
r-+e--e-e _
-By 0 0
30
I 60
I 90
120
150
_ 180
-210 240
270
Q 308
(min)
FIGURE 1. Enzyme release and mechanical activity in the hearts treated or not treated for cardioplegia. Changes in enzyme releases (MDH, LDH and CPK), perfusion pressure (PP) and heart rate (HR) were examined over 5 h. Vertical bars indicate means f S.E.M. (0) Cardioplegia (n = 13); (0) Non-treated (n = 6).
Tyrode solution. Following perfusion with warm Tyrode solution, contractions began at 23 f 2 s. The initial enzyme release following perfusion stabilized within 30 min. Heart rate remained constant throughout the study, but perfusion pressure rose progressively. The treated hearts usually stopped beating in the diastole at 5.6 & 0.3 s (rr = 46) following the injection of cardioplegic solution. Strong contractions began at 54 f 4 s following exposure to warm Tyrode solution. Stable levels of enzyme release and mechanical activity were reached within 30 to 60 min after the beginning of perfusion, and were maintained for at least 5 h. Concerning each enzyme release there were no systematic differences between the groups treated and not treated for cardioplegia. However, higher levels in heart rate and perfusion pressure were observed in the non-treated group. Perfusate selection
The treated hearts were perfused with Krebs-Henseleit, Locke and Tyrode solutions, respectively (Table 1). Figure 2 shows the enzyme activities in the effluent fluid and mechanical activities.
832
K. SAKAI
500
-T
ET AL.
MDH
400 300 200 100 7 i 2 E
0 200
LDH
-T
100 0 400 300 200 100 0
Tc 5
HR
150 100 50
PP 2
60
+-.--o-o-o-(
1
30 0
@q+-^e~: I I 30 60
0
I 90
I 120
I 150
I I8
(min)
FIGURE 2. Enzyme release and mechanical activity in the heart preparations perfused with Krebs-Henseleit, Locke and Tyrode solutions, respectively. The hearts were subjected to cardioplegic treatment. Changes in enzyme releases (MDH, LDH and CPK), perfusion pressure (PP) and heart rate (HR) were examined over 3 h. Vertical bars indicate means 6 S.E.M. (0-O) Locke (R = 4); (H) Krebs-Henseleit (n = 9); (@- - - -0) Tyrode (n = 13).
Even after 30 min of perfusion using Krebs-Henseleit solution considerable enzyme release was observed, with the exception of LDH. Although a steady state level of MDH and CPK release was obtained about 90 min after the start of perfusion, the enzyme levels tended to rise again. Perfusion pressure and heart rate also rose gradually. Perfusion with Locke solution induced more pronounced enzyme release than with Krebs-Henseleit solution. Ninety minutes after starting the perfusion a steady state
ENZYME
RELEASE
AND
MYOCARDIAL
833
ISCHEML4
of enzyme release was obtained at relatively high level. Progressive rise in perfusion pressure and heart rate was also observed. It was difficult to maintain the pH of 7.4 constantly during the perfusion. A steady level of enzyme release was reached later with Krebs-Henseleit or Locke solution than with Tyyrode solution. It might be finally concluded that a stable level of both enzyme release and mechanical activity was more rapidly arrived at with Tyrode perfusion, and it was therefore more suitable for the present study.
400,
300 c%s
I MDH
I
LDH --o
HR
50 100
50 0
PP 0
30
60
SO 120 150 160 210 240 270 300
FIGURE 3. Effects of variations inthe glucose concentration of Tyrode solution on enzyme release and mechanical activity. Glucose was added in the solution at 4, 6 and 12 mre concentrations. Changes in enzyme release (MDH, LDH and CPK), perfusion pressure (PP) and heart rate (HR) were examined over 5 h. Vertical bars indicate means f S.E.M. (ha) 4 mr+ Glucose (a = 4) ; (@- - +) 6 m~/l Glucose (a = 13); (O-0) 12 mM/l Glucose (a = 4).
Tyrode solution was modified by the addition of different concentrations of glucose. Figure 3 summarizes the effect of alterations in the glucose concentrations (4, 6 and 12 mM) of perfusate on enzyme release and mechanical activity. After the beginning of perfusion, the perfusate containing 12 mM glucose induced more marked enzyme release (especially MDH and CPK) than those with 4 and 6 mM glucose, and did not rapidly produce a steady level. Even after 1 h of perfusion, the values for MDH and CPK release at 12 mM glucose were significantly higher than those at 4 and 6 mM glucose (P < 0.001). Although changes in enzyme
834
K. SAKAI
ETAL.
release after perfusion with 4 and 6 mM glucose followed a similar pattern, a more stable level seemed to be induced by that with 6 mM glucose. In all cases, perfusion pressure and heart rate remained at a constant level throughout the study.
Pre-ischemia
(60
Pump stop
Reperfusion
i
i Post-ischemia
lschemia
min )
w’)I min u-1
I t t Control (Sampling)
t Onset of perfusion
’
I
I’
I
I
I
I
I
I
2’
3’
4’
5’
6’
7’
L
Sampling
t
FIGURE 4. Diagram of perfusion plan under the ischemic experiments.
Enzyme releasefollowing total ischemia The effect of cardioplegia on enzyme release was tested in experiments with 20 min ischemia. After a 60 min equilibration period hearts were subjected to total ischemia by stopping the perfusion pump (Figure 4). Following reperfusion aliquotes were TABLE
3. Appearance reperfusion
Duration of &hernia Incidence of arrest Per cent Begin of recontraction
of cardiac arrest following
(min)
(s)
ischemia and recontraction
following
5
10
20
4113 31
516 83 39.0 5 2.9*
616
30.7 f 1.6*
100
46.7 + 5.6
Hearts not treated for cardioplegia were subjected to total ischemia of different periods. Mean time interval for the appearance of cardiac arrest following initiation of ischemia; 6.9 & 2.6 min (n = 15). The values marked with asterisk were calculated only for the preparations, in which cardiac arrest occurred after ischemia. Values represent means 5 S.E.M. These values differ significantly with P < 0.001 (H-test). continuously collected and sampled at the following intervals: 30”, 1’) 2’, 3’, 4’, 5’, 6’, 7’. The sample containers were replaced by clean, fresh tubes at the termination of each interval. The activities represent the integral enzyme releases for the successive intervals. As observed in Figure 5 and Table 4, the hearts treated with cardioplegic solution showed significantly less enzyme release compared with the non-treated. The cardioplegic solution was not used in a series of ischemic studies, because it possessed some influence on enzyme release even after 1 h of perfusion.
ENZYME
RELEASE
AND
MYOCARDIAL
MDH
0 Cardioplegia 0 Non-treated
0
I
2
3 (min)
4
835
ISCHEMIA
5
(n=6) (n:6)
6
7
FIGURE 5. Myocardial enzyme release after 20 min of total &hernia. After 60 mm-equilibration period total ischemia was induced by stopping perfusion pump. Vertical bars indicate means & S.&M. for enzyme amount released following reperfusion. Each value was expressed in per cent increase over pm-ischemia control. The maximum values (first phase) of individual enzymes (MDH, LDH and CPK) were significantly different from the corresponding control values (P < 0.001). Two minutes after reperfusion (second phase), only each value of LDH and CPK in the hearts not treated for cardioplegia was significantly different from the corresponding control value (P < 0.1) ; (0) Cardioplegia (a = 6); (0) Non-treated (n = 6).
Table 4 indicates the results of enzyme release for various durations of total ischemia: 5, 10 and 20 min. The releases generally occurred in a biphasic pattern: a marked release immediately after reperfusion (first phase) and later a moderate release (second phase) (Figure 5). In the first phase the maximum release of MDH, LDH and CPK occurred within 30 s following reperfusion. During this time the resumption of contraction was not yet observed (Table 3). The releases were transient and the level returned close to the control levels within 60 s; after that a
836 TABLE
K. SAUAI ETAL.
4. Myocardial
enzyme release after different ischemic durations Cardioplegia
No cardioplegia Duration of &hernia (min) ~--
5 (n = 13)
20 (n = 6)
10 (n = 6)
MDH LDH CPK
225.7 f 18.1 240.0 & 23.4 274.7 f 30.0
Maximum 581.4 5 115.1 449.2 & 33.2 628.5 f 72.7 429.0 + 50.4 711.8 5 117.6 438.8 & 51.7
MDH LDH CPK
299.0 f 34.3 350.7 f 50.4 313.2 f 53.3
584.7 & 30.1 560.8 f 54.6 481.0 f 99.1
20 (n = 6) 310.6 + 75.6 150.8 & 18.7 233.2 & 10.0
Total 798.8 & 72.3 1195.8 f 195.0 1035.8 f 310.9
411.2 + 93.0* 279.0 f 56.4** 322.2 f 18.4**
Each value was expressed in per cent increase over the control value. Maximum : maximum enzyme release in the first phase. Total: total enzyme release into the effluent perfusate from the hearts during 5 min after reperfusion. Each value is mean & S.E.M. Values marked with asterisks in the table were significantly different (u-test) from those of the corresponding 20 min ischemia group not treated for cardioplegia.* P < 0.025;** :P < 0.005. Total values in the group not treated for cardioplegia differ significantly from the other values in this group with P < 0.01 (MDH), P < 0.01 (LDH) and P < 0.05 (CPK), respectively (H-test). moderate release (second phase), always accompanied by the resumption of contraction, lasted for a few minutes. Even if the hearts were subjected to 20 min of total ischemia, lactate output and enzyme release as well as mechanical activity returned close to the control levels about 7 min after reperfusion (Table 5). The rate of each enzyme release and the time contraction began for all ischemic periods tested was dependent on the duration of ischemia, as shown in Tables 3 and 4. TABLE
5. Myocardial
metabolic changes before/after 20 min ischemia Time after reperfusion (min)
7 Lactate formed (pmol/min 0.20 & 0.03 0.26 f 0.06 Control
6 MDH released (mU/min 32.4 f 3.4 48.2 -& 7.8
15 / g) 0.20 f 0.03 14
Control
/ g) 39.3 & 6.4
Following reperfusion aliquots were collected for 1 min at each time interval. The amounts of lactate and MDH were expressed in terms of gram wet heart weight before perfusion (see methods). The values in this table represent means & S.E.M. for, respectively, four hearts not treated for cardioplegia. These values were not significantly different from the control means (P > 0.05) (U-test). Each control value was obtained within 2 min prior to ischemia. Perfusion pressure: control, 20.8 f 1.3 mmHg; min after reperfusion, 21.5 5 1.5 mmHg (P > 0.05). Heart rate: control, 154.0 f 4.8 min- 1; 7 min after reperfusion, 158.5 f 7.9 min-l (P > 0.05).
ENZYME
RELEASE
AND
MYOCARDIAL
ISCHEMIA
837
4. Discussion The present results show that brief, total myocardial ischemia induces pronounced but transient enzyme release from the myocardium. The release need not necessarily be related to irreversible cell damage. Prior to the ischemic studies it was important to establish the most suitable experimental conditions. Shipp et al. [20], using isolated working rat heart, have reported that perfusion rates of 5 ml/min or higher are needed to avoid metabolic changes suggestive of oxygen deficiency. The present study, however, showed that the different coronary flow rates ranging from 4 to 8 ml/min induced no significant differences on the rates of lactate output. These values were in accordance with those calculated from another study [1.5] at a comparable flow rate. According to Fisher and Williamson using rats of 200 to 260 g [5], there was an adequate oxygen supply at 4 ml/min perfusion rate with oxygen saturated solution and further elevation of oxygen extraction with 2,4-dinitrophenol. It was tried to minimize the myocardial energy demand during the operation, not only by decreasing myocardial temperature but also by using the cardioplegic solution [2]. The myocardial oxygen deficiency, which occurs during the operation, might induce the release of enzymes. The myocardial oxygen demand is reduced from the normal heart condition by a factor of about 100 when the cardioplegic solution is used at 5°C. In fact, a more stable condition in the preparations treated with the cardioplegic solution was maintained over a longer period in comparison with the non-treated. Effects of several perfusates (Tyrode, Krebs-Henseleit and Locke solution) were compared in the hearts treated with the cardioplegic solution. Tyrode solution had the most suitable effect on enzyme release as well as on mechanical activity, which has been shown to be an indication of myocardial energy demand [12]. Tyrode solution maintained a stable state of the preparation for at least 5 h (Figure 1). Several workers have reported protective effects of glucose on ischemic and/or anoxic hearts [lo, 161. Therefore, the modification in the concentration of glucose in the perfusate might be interesting. As observed, a more stable level seemed to be produced by the perfusion with 6 mM glucose (Figure 3). Under 20 min ischemia, the induced enzyme releases for the group treated for cardioplegia were smaller than for the non-treated group. This finding can be explained by the reduction of myocardial oxygen demand due to cardioplegia and/or a membrane stabilizing effect of procaine. Under clinical conditions, procaine in a cardioplegic solution will be rapidly inactivated by blood esterases [17], whereas in the present study it seems to decrease membrane permeability to myocardial enzymes by close binding with the cell membrane over a long period. Under different durations of total ischemia, enzyme release generally occurred in a biphasic pattern; a marked release just after reperfusion (first phase) and later a moderate release (second phase). The first phase of release occurred without the
838
K. SAKAI
ETAL.
concomitant resumption of contraction. De Leiris et al. [3] have stated that cardiac contraction is a major determinant of enzyme release into the perfusion fluid. However, under the present ischemic conditions, the resumption of contractility was not necessarily needed for enzyme release. In clinical practice it is believed that elevations of serum enzymes following myocardial &hernia are related quantitatively to enzyme release from irreversibly injured cells [22]. However, the mechanism of enzyme release is still controversial. Hearse et al. [7] have stated that cellular leakage of enzymes occurs when the cell structure is damaged. It has also been quoted by De Leiris et al. [3] that enzyme release entails an increase of membrane permeability. Zierler [27] also suggested that enzymes could be released through an alteration of membrane permeability under some conditions. According to our present results, the increased enzyme release appearing after total ischemia for the tested durations might not necessarily result from irreversible cell damage, but rather from a transient increase in membrane permeability to myocardial enzymes during/after ischemia. The relatively rapid recovery of mechanical activity as well as enzyme release and lactate output after 20 min of total ischemia definitely suggests this possibility, although Hearse and Stewart [8], using isolated working rat hearts subjected to a comparable ischemic period, deduced opposite conclusions. Despite some differences in methods and species, our present views would be supported by the conclusions of Neely et al. [13] and others [23] in which permanent myocardial damage did not occur if the duration of ischemia was less than 20 min. Experimental and clinical findings would apparently seem to lend further support to our views. It is known that changes in coronary arterio-venous differences in enzymes occur within 20 to 30 min after the onset of local oxygen deficiency in the myocardium [19, 261. As previously reported, there is a close correlation between the rate of enzyme release and myocardial high energy phosphate content [24] ; appreciable amounts of enzyme proteins released from myocardial cells into the coronary effluent were observed after only a small decrease of myocardial ATP. At this stage myocardial cells did not yet sustain irreversible damage. The more the myocardial ATP content decreased, the more the rate of enzyme release increased. The results presented in Table 4 might partly be accounted for by the above description. It remains to be studied whether there is a direct causal relationship between changes in energy metabolism and the liberation of enzymes. Acknowledgments We are very grateful to Mrs R. Hahn, Mrs G. Sander and Miss A. Holtmeyer for their excellent assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 89, Kardiologie Gottingen). A part of this study will be presented as an inaugural dissertation of the University of Gottingen by M. M. Gebhard.
ENZYME RELEASE AND MYOCARDIAL
ISCHEMIA
839
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