Effects of dexamethasone the early phase of acute
on myocardial cells in myocardial infarction
James A. Spath, Jr., Ph.D.* Allan M. Lefer, Ph.D** Philadelphia,
Pa.
Libby and co-workers’ have shown that pharmacologic doses of cortisol limit infarct size 24 hours after coronary artery occlusion in dogs. Although the mechanism of the beneficial action of glucocorticoids upon the myocardium is unclear, several mechanisms could play a role in their protective effect on the myocardium. These mechanisms are: coronary vasodilation, improved myocardial contractility, and stabilization of cellular membranes within the ischemic myocardium. The membrane-stabilizing action of the drug would be reflected by a diminished loss of myocardial cellular enzymes from the ischemic portion of the myocardium. Since lysosomal hydrolases have been implicated in the propagation of the cellular injury during the early stages of acute myocardial infarction,* this action may be crucial in protecting the heart against ischemic damage. Moreover, glucocorticoids are very effective in stabilizing lysosomal membranes. Recently, we demonstrated such an effect for the glucocorticoid, methylprednisolone.3 Since methylprednisolone and dexamethasone have been shown to have similar beneficial effects in other conditions of circulatory dysfunction,“. 5 the present study was undertaken to investigate whether From the Department of Physiology, University of Virginia School of Medicine, Charlottesville, Va. and the Department of Physiology, Jefferson Medical College of Thomas Jefferson University, Philadelphia. Supported National
by National Institutes of Health Heart and Lung Institute.
Received
for publication
Reprint requests: Jefferson Medical St., Philadelphia, *Special National
July
Grant
HL-14277
from
the
22, 1974.
Dr. James A. Spath, Jr., Department College, Thomas Jefferson University, Pa. 19107.
National Institutes of Health Heart and Lung Institute.
**Present address: Department of College, Thomas Jefferson University, Pa. 19107.
Postdoctoral Physiology, 1020 Locust
of Physiology, 1020 Locust Fellow
of the
Jefferson Medical St., Philadelphia,
pre- or posttreatment with dexamethasone alters myocardial enzyme release or electrocardiographic signs of myocardial damage during acute myocardial &hernia. Method Coronary artery occlusion. Twenty-one male and female cats (2.3 to 3.6 kilograms) were anesthetized with sodium pentobarbital (30 mg. per kilogram) given intravenously. The trachea was cannulated and positive-pressure respiration was instituted with a Harvard respirator. Catheters were placed within the right external jugular vein and left common carotid artery and positioned for the recording of central venous pressure (CVP) and mean arterial blood pressure (MABP), respectively. Intravascular pressures were recorded continuously using appropriate Statham P-23 pressure transducers coupled to a Beckman Type R Dynograph. Needle electrodes were placed subcutaneously to allow continuous recording of Lead III of the electrocardiogram (ECG). A midsternal thoracotomy was performed, the heart exposed, and a noncannulating electromagnetic flow probe placed around the root of the aorta. The output of the flow probe was amplified by a Statham Model 4001 flowmeter and continuously recorded on the oscillographic recorder. The left coronary artery was cleared of surrounding tissue and a 3-O silk ligature was placed under the vessel. In cats subjected to acute myocardial ischemia, the ligature was tied tightly around the left coronary artery 13 to 15 mm. from the coronary ostium. Cats were either sham-operated or subjected to five hours of myocardial ischemia (MI) following occlusion of the coronary artery. Cats subjected to MI, were given either dexamethasone sodium phosphate (Decadron, Merck) 8 mg. per kilogram or an equal volume of vehicle in
July,
1975,
Vol.
90, No.
1, pp.
50-55
Effects
the jugular vein catheter. Dexamethasone was administered slowly over ten minutes beginning either 30 minutes prior to, or 60 minutes following, occlusion of the coronary artery. Shamoperated cats were given dexamethasone 30 minutes prior to the start of the experiment.
of dexamethasone on m,vwordial
Hemodynamic
Effects ligation
of Coronary
cells
Artery
Sampling and homogenization of cardiac tissue. Samples of arterial blood (4 ml.) were
withdrawn from the carotid catheter just prior to occlusion and at one, two, four, and five hours after occlusion. Blood samples were drawn from sham-operated cats at the same time. Blood loss was replaced with an equal volume of KrebsHenseleit solution warmed to 37” C. Blood was collected in polyethylene tubes containing two drops of sodium heparin, 1,000 units per milliliter (Upjohn, beef lung) and centrifuged at 2,400 x g and 4” C. for 15 minutes. The plasma was decanted and treated as described below. At five hours, the hearts were excised, rinsed in 0.9 per cent NaCl solution at 4” C., rapidly weighed, and placed in cold 0.25 M sucrose. The heart was divided into ischemic and normal left ventricle by inspection of the coronary vessels, endocardium, and epicardium. Thus, tissue supplied by arterial branches distal to the ligature appeared as a cyanotic area having patchy subendocardial hemorrhagic regions. Ischemic or nonischemic tissue was homogenized in 0.25 M sucrose (l:lO, w:v) containing 1 mM ethylenediaminetetraacetic acid and 0.1 mM mercaptoethanol for the determination of myocardial creatine phosphokinase (CPK) activity.” The tissue preparations for the CPK determinations were treated according to the method of Kjekshus and Sobel.7 Additional ischemic or adjacent normal ventricular tissue was minced and homogenized in 0.25 M sucrose (l:lO, w:v) for subsequent determination of lysosomal enzymes. Each sample of myocardium was homogenized twice for 15 seconds using a Virtis homogenizer at a speed setting of 9.0. The homogenates were centrifuged at 800 x g for 10 minutes. The supernatants were centrifuged at 36,000 x g for 30 minutes at 4O C. Supernatants were assayed for /?-glucuronidase, and cathepsin D activities in the presence and absence of Triton X-100. Cardiac homogenates from sham-operated cats were derived from left ventricular tissue anatomically equivalent to the ischemic and normal areas present in ischemic hearts. Biochemical determinations. Aliquots of the
American Heart Journal
0
1
2
lime
3
4
5
(hours)
Fig. 1. Hemodynamic effects of coronary artery ligation in cats receiving the vehicle for dexamethasone 30 minutes prior to ligation. Ligation of the left coronary artery 13 to 16 mm. from the coronaql ostium produced significant decreases in the heart rate, mean arterial blood pressure, and aortic blood flow. Values are means k S.E.M. for four cats.
plasma and supernatants of cardiac homogenates were analyzed for activities of the lysosomal protease, cathepsin D, and of another lysosomal hydrolase, P-glucuronidase, using the method of Anson* and Talalay, Fishman, and HugginsQ respectively. The protein concentration of plasma and cardiac tissue supernatants was determined using the biuret method.“’ Cathepsin D activity is expressed as milliequivalents of tyrosine x lo-” released from bovine hemoglobin per milligram of protein per hour at 37” C. Plasma and cardiac CPK activities were determined according to the method of Rosalki.” CPK activity is expressed as international units per milligram of protein. One international unit of CPK is that activity which transfers 1.0 pmole of phosphate from phosphocreatine to ADP per minute at pH 7.4 at 30” C. Results
Fig. 1 illustrates the heart rate, mean arterial blood pressure, and aortic blood flow just prior to, and for five hours following coronary ligation in untreated cats (i.e., cats receiving the vehicle for dexamethasone). Ligation of the coronary artery
51
Spath and Lefer
-Ml+V.hi.l.
TIME (hours)
2. Effect of dexamethasone (Dexa) or its vehicle upon the elevation of the S-T-segment in cats subjected to acute myocardial &hernia (MI) by occlusion of the left coronary artery. All values are means f S.E.M. for four to six cats in each group. Sham-operated cats given Dexa showed an absence of S-T-segment elevation over the entire five-hour experimental period. Occlusion of the left coronary artery initially produced similar elevations of the S-T-segment in all three groups of occluded cats. The S-T-segment of vehicletreated cats (MI + vehicle) remained elevated for the duration of the experiment. However, S-T-segment elevation declined toward control values five hours after occlusion in MI cats posttreated with Dexa (MI + Dexa, post). mV = millivolts. Fig.
produced moderate but statistically significant (p < 0.02) decreases in heart rate, mean arterial blood pressure, and aortic flow within one hour. Furthermore, the hemodynamic response of ligated cats receiving dexamethasone did not differ from that of the animals given vehicle. In contrast to cats experiencing coronary artery ligation, sham-operated cats maintained stable normal values for heart rate, blood pressure, and aortic flow over the five-hour experimental period. Furthermore, the directly measured central venous pressures and the calculated total peripheral resistances of untreated cats were similar to dexamethasone-treated cats throughout the postocclusion period. Thus, we do not find any overt hemodynamic action of dexamethasone in our experiments with regard to systemic vasodilator or positive inotropic effects. In four cats, ventricular fibrillation occurred spontaneously 20 to 30 minutes after coronary artery ligation. Cats experiencing a single episode of ventricular fibrillation were rapidly converted to sinus rhythm using a Medtronic Internal-
52
External defibrillator (Minneapolis, Minn.). For the remainder of the experimental period, these cats displayed mean hemodynamic parameters and loss of myocardial enzymes which were similar to the majority of cats without ventricular fibrillation. Cats in which the ventricle fibrillated a second time were discarded. The S-T-segment elevation recorded in dexamethasone-treated and untreated cats is shown in Fig. 2. Sham-operated cats given dexamethasone exhibited no significant change in S-T-segment elevation during five hours of observation. In contrast, marked elevation of the S-T-segment occurred at 20 to 60 minutes in all groups of cats subjected to coronary artery occlusion. Moreover, S-T-segment remained elevated for the entire experimental period in untreated cats and in cats pretreated with dexamethasone. However, posttreatment of ischemic cats with dexamethasone was associated with a significant reduction in the S-T-segment five hours after coronary artery occlusion. Concomitant with the observed electrocardiographic changes, increases in plasma CPK activities occurred in all experimental groups (Fig. 3). A significant elevation of plasma CPK activity occurred as early as two hours following coronary artery occlusion in untreated cats. Plasma CPK activity continued to increase, reaching a value of eight times the initial plasma CPK activity five hours after coronary artery occlusion. In contrast, sham-operated cats exhibited only a moderate increase in plasma CPK activity after five hours. Moreover, both pre- and posttreatment of cats with dexamethasone limited the increase in plasma CPK activity so that five hours after coronary artery occlusion, plasma CPK activity was not significantly different from sham-operated control animals. Although plasma CPK activities were altered by MI, the plasma activities of /3-glucuronidase and cathepsin D remained unchanged for the fivehour experimental period. The absence of increased plasma lysosomal hydrolase activities following myocardial ischemia correlated well with the relatively well maintained mean arterial blood pressure and aortic flow in coronary-ligated cats (Fig. 1). Adequate tissue perfusion in peripheral organs prevented loss of lysosomal hydrolases from noncardiac tissues. In contrast to the plasma activities of the lysosomal hydrolases, /3-glucuronidase and ca-
July, 1975, Vol. 90, No. 1
Elpects
thepsin D, the myocardial activities of these enzymes were affected by coronary artery ligation. Fig. 4 summarizes the myocardial cathepsin D activities of normal and ischemic myocardial tissue obtained from dexamethasone-treated and untreated cats. In cats subjected to sham MI (i.e., coronary artery isolated but not ligated) normal tissue and tissue which would have become ischemic if the coronary artery had been ligated, had very similar activities of the lysosomal protease, cathepsin D, (i.e., about 20 units per milligram of protein). Futhermore, nonischemic myocardial tissue of hearts subjected to coronary ligation also showed a cathepsin D activity approximating 20 units per milligram of protein. However, ischemic myocardial tissue of cats given the steroid vehicle exhibited a 40 per cent reduction in cathepsin D activity compared to the activity of adjacent normal tissue or to left ventricular tissue excised from sham-operated cats. In contrast, ischemic myocardial tissue of cats given dexamethasone one hour after coronary artery ligation exhibited a cathepsin D activity similar to that of normal tissue, 23.9 + 2.1 units vs. 20.7 rtr 1.4 units, respectively. However, pretreatment with dexamethasone appeared less effective in preventing the loss of myocardial cathepsin D. Thus, ischemic tissue of pretreated cats had 16 per cent less protease activity when compared to adjacent, normal tissue (p< 0.02). The alterations in myocardial CPK activities (Fig. 5) were very similar to those of cardiac cathepsin D and reflected the changes found in plasma CPK activities in the four experimental groups. Thus, the CPK activity of ischemic tissue of untreated MI cats was 40 per cent lower than the normal ventricular tissue of these hearts. Furthermore, posttreatment of MI cats with dexamethasone significantly prevented the loss of enzyme activity from the ischemic myocardium. However, pretreatment with dexamethasone was significantly less effective than posttreatment in preventing loss of CPK activity from the ischemic portion of the myocardium. Ischemic tissue of pretreated cats had 30 per cent less enzyme activity than adjacent normal tissue, a level of enzyme activity not significantly different from that observed in ischemic tissue of untreated cats. Assay of the cardiac activities of the lysosomal hydrolase /3-glucuronidase in normal and ischemic myocardium presented a similar pattern
American
Heart
Journal
of dexamethasone
on mywurdial
cells
,.*;
0.6
-
0.4
-
0.2
-
01
’
1
I
I
I
I
0
1
2
3
4
5
Time
(hours)
Fig. 3. Effect of dexamethasone or its vehicle upon plasma CPK activity before and after induction of acute myocardial &hernia (MI). All values are means rt S.E.M. expressed in International Units (IU) X lo-’ per milligram of protein. Plasma CPK activity increased eightfold in cats given vehicle prior to occlusion. A significant increase in plasma CPK occurred as early as two hours after occlusion. Treatment of cats with dexamethasone 30 minutes before or 60 minutes after occlusion prevented much of the increase in plasma CPK activity following MI.
to that observed for cardiac cathepsin D and CPK activities. Discussion
Ligation of the coronary artery produced moderate decreases in heart rate, mean arterial blood pressure, and aortic flow (Fig. 1). However, pharmacologic doses of dexamethasone given either before or after coronary artery ligation did not alter this hemodynamic pattern from that of cats given only the vehicle for dexamethasone (i.e., sterile water containing the steroid preservatives). These data indicate that dexamethasone does not alter the hemodynamic response to &hernia at least within the first five hours following coronary artery occlusion. Thus, we do not find any overt vasodilating or positive inotropic effect of dexamethasone in these experiments. This lack of a hemodynamic effect is in agreement with earlier findings’.” in dogs and in catsI wherein pharmacologic doses of dexameth-
53
Spath
and
Lefer
Myocardial
.-s
0 z h m
< * C 5
Cathepsin
BQ
Normal
m
Irchemic
D Activity
Myocardial
Myocordium
m
Myocardium
CPK
Activity
Normal
Myocordium
lschemic
Myocardium
20 24 2o 16 12 0 4 0 LSham D&l
MIA
MI V&l*
MI Df, beI
MI D&Cl (P-t)
L Sham D&a
MI _T
MI
MI
V&cl*
D&i (Pre)
Fig. 4. Effect of pre- or posttreatment with dexamethasone upon the cathepsin D activity of normal or ischemic myocardium. In cats given vehicle, myocardial ischemia resulted in 40 per cent decrease in the cathepsin D activity of ischemic ventricle relative to adjacent, normal myocardium. Pretreatment with dexamethasone reduced the loss of cathepsin D activity to 16 per cent within ischemic myocardium. Posttreatment with glucocorticoid prevented the loss of lysosomal hydrolases in acute myocardial &hernia. Values are means f S.E.M. for the number of samples shown on each bar.
Fig. 5. Effect of pre- or posttreatment with dexamethasone upon the myocardial CPK activity of normal or ischemic myocardium. All values are means + S.E.M. Numbers at the bottom of each bar indicate the number of myocardial samples studied. Ischemic myocardial tissue obtained from vehicle-treated cats (MI + vehicle) shows a 40 per cent decrease when compared with adjacent normal left ventricle. Posttreatment of cats with dexamethasone prevented the decrease in the CPK activity of ischemic heart tissue.
asone and other glucocorticoids failed to exhibit significant vascular or inotropic effects. The similarity of the hemodynamic response of ligated cats was also reflected in the early electrocardiographic changes evident in these cats. Marked elevation of the S-T-segment occurred at 20 to 60 minutes in all groups of cats subjected to coronary artery ligation indicating comparable degrees of initial myocardial injury.13 Although cats given the vehicle for dexamethasone or pretreated with the glucocorticoid maintained similarly elevated S-T-segment voltage after five hours, posttreatment of cats with dexamethasone was associated with a reduction in the S-Tsegment elevation at that time. Thus, administration of dexamethasone one hour after coronary artery ligation had a beneficial effect as assessed by the reduction of the S-T-segment elevation in the early phase of acute myocardial ischemia. The ineffectiveness of pretreatment with dexamethasone in reducing the S-T-segment elevation may be related to the plasma and tissue levels of the glucocorticoid at the time when ischemic myocardial cells began to undergo irreversible damage (i.e., about two hours after occlusion).”
Thus, pretreatment with dexamethasone results in degradation of the steroid so that inadequate concentrations are present within the ischemic portion of the myocardium at the critical twohour period. In contrast, posttreatment with dexamethasone provides a high level of glucocorticoid two hours after occlusion, the time at which the rate of cellular damage is increasing as indicated by the steep rise in plasma CPK activity of untreated cats. Determination of myocardial CPK activity of normal and ischemic tissue indicated that the observed alterations in plasma CPK activity in MI cats actually reflected loss of myocardial CPK. These findings also suggest that posttreatment with dexamethasone was more effective than pretreatment in preventing loss of CPK activity from ischemic tissue. Similar results were obtained with regard to myocardial cathepsin D activity. Dexamethasone, given after coronary artery ligation also prevented the loss of cathepsin D activity from ischemic myocardial tissue more effectively than pretreatment with the same dose of dexamethasone. The 40 per cent loss of myocardial lysosomal
54
July, 1975, Vol. 90, No. 1
Effects of dexamethasone
hydrolase activity within ischemic myocardium agrees closely with the findings of Ricciutti2,15 in dogs, not only in quantity but in rate of loss. Thus, Ricciutti measured comparable loss of lysosomal hydrolase activity within four hours after coronary artery occlusion. Our results indicate that the loss of myocardial lysosomal hydrolase activity within ischemic myocardium can be significantly reduced by administration of pharmacologic doses of dexamethasone. Furthermore, these results are consistent with the hypothesis that dexamethasone stabilizes myocardial cellular or subcellular membranes within the ischemic myocardium or at the border of the evolving infarct. The membrane-stabilizing action of the glucocorticoid suggests a mechanism for the infarct-reducing effect of other glucocorticoids reported previously.‘, .i
Dexamethasone exerted no significant hemodynamic effect in sham-operated cats or in cats subjected to acute myocardial &hernia. However, the glucocorticoid did normalize elevated S-T-segments toward pre-ischemic values, and prevented much of the increase in plasma CPK activity following coronary artery ligation. Moreover, dexamethasone prevented loss of CPK activity and restricted the loss of lysosomal hydrolase within ischemic myocardial tissue. These data indicate that lysosomal disruption is an early consequence of myocardial ischemia and that treatment with dexamethasone prevents the loss of myocardial lysosomal and cellular enzymes as reflected in normalization of the ECG and plasma CPK activity of ischemic cats. In this way, dexamethasone may act to retard the spread of the developing infarct within the ischemic myocardium. The authors wish to acknowledge assistance of Mrs. Ann Gibb and Mr.
American
Heart
Journal
cells
the course of this investigation. We are also grateful for the generous gift of dexamethasone from Dr. Ingeborg Schultze of the Merck Institute of Therapeutic Research, West Point, Pa. REFERENCES 1.
2. 3.
4.
5.
6.
7.
Summary
on m,wcardial
8.
9.
10.
11.
12.
13.
14. 15.
Libby, P., Maroko, P. R., Bloor, C. M., Sobel, B. E., and Braunwald, E.: Reduction of experimental myocardial infarct size by corticosteroid administration, J. Clin. Invest. 52:599, 1973. Ricciutti, M. A.: Lysosome and myoc,ardial cellular injury, Am. J. Cardiol. 30:498, 1972. Spath, J. A., Jr., Lane, D. L., and Lefer, A. M.: Protective action of methylprednisolone on the myocardium during experimental myocardial ischemia in the> cat, Circ. Res. 35:44, 1974. Spath, J. A., Jr., Gorczynski, R. J., and Lefer, A. M.: Possible mechanisms of the beneficial action of glucocorticoids in circulatory shock, Surg. Gvnecol. Obstet. 137:597, 1973. Lefer, A. M., and Martin, J.: Mechanism of the protective effect of corticosteroids in hemorrhagic shock, Am. J. Physiol. 216:314, 1969. Rosalki, S. B.: An improved procedure for creatine phosphokinase determination, J. Lab. Clin. Med. 69:696, 1967. Kjekshus, J. K., and Sobel, B. E.: Depressed myocardial creatine phosphokinase activity following experimental myocardial infarction in rabbit, Circ. Res. 27:403, 1970. Anson, M. L.: The estimation of cathepsin with hemoglobin and the partial purification of cathepsin, J. Gen. Physiol. 20:565, 1936. Talalay, P., Fishman, W. H., and Huggins, C.: Chromogenic substrates. II. Phenolphthalein glucuronic acid as substrate for the assay of glucuronidase activity, J. Biol. Chem. 166:757, 1946. Gornall, A. G., Bardowill, C. J., and David, M. M.: Determination of serum proteins by means of the biuret reaction, J. Biol. Chem. 177:751, 1949. Gorczynski, R. J., Spath, J. A., Jr., and Lefer, A. M.: Vascular responsiveness of the in situ perfused dog pancreas, Eur. J. Pharmacol. 27:68, 1974. Kadowitz, P. J., and Yard, A. C.: Circulatory effects of hydrocortisone and protection against endotoxin shock in cats, Eur. J. Pharmacol. 9:311, 1970. Shell, W. E., Lavelle, J. F., Covell, J. W., and Sobel, B. E.: Early estimation of myocardial damage in conscious dogs and patients with evolving acute myocardial infarction, J. Clin. Invest. 52:2579, 1973. Brachfeld, N.: Maintenance of cell viability, Circ. 39:(Suppl. 4), 202, 1969. Ricciutti, M. A.: Myocardial lysosome stability in the early stages of acute ischemic injury, Am. d. Cardiol. 30:492, 1972.
the expert technical Robert Stickley during
55
on myocardial cells in myocardial infarction
James A. Spath, Jr., Ph.D.* Allan M. Lefer, Ph.D** Philadelphia,
Pa.
Libby and co-workers’ have shown that pharmacologic doses of cortisol limit infarct size 24 hours after coronary artery occlusion in dogs. Although the mechanism of the beneficial action of glucocorticoids upon the myocardium is unclear, several mechanisms could play a role in their protective effect on the myocardium. These mechanisms are: coronary vasodilation, improved myocardial contractility, and stabilization of cellular membranes within the ischemic myocardium. The membrane-stabilizing action of the drug would be reflected by a diminished loss of myocardial cellular enzymes from the ischemic portion of the myocardium. Since lysosomal hydrolases have been implicated in the propagation of the cellular injury during the early stages of acute myocardial infarction,* this action may be crucial in protecting the heart against ischemic damage. Moreover, glucocorticoids are very effective in stabilizing lysosomal membranes. Recently, we demonstrated such an effect for the glucocorticoid, methylprednisolone.3 Since methylprednisolone and dexamethasone have been shown to have similar beneficial effects in other conditions of circulatory dysfunction,“. 5 the present study was undertaken to investigate whether From the Department of Physiology, University of Virginia School of Medicine, Charlottesville, Va. and the Department of Physiology, Jefferson Medical College of Thomas Jefferson University, Philadelphia. Supported National
by National Institutes of Health Heart and Lung Institute.
Received
for publication
Reprint requests: Jefferson Medical St., Philadelphia, *Special National
July
Grant
HL-14277
from
the
22, 1974.
Dr. James A. Spath, Jr., Department College, Thomas Jefferson University, Pa. 19107.
National Institutes of Health Heart and Lung Institute.
**Present address: Department of College, Thomas Jefferson University, Pa. 19107.
Postdoctoral Physiology, 1020 Locust
of Physiology, 1020 Locust Fellow
of the
Jefferson Medical St., Philadelphia,
pre- or posttreatment with dexamethasone alters myocardial enzyme release or electrocardiographic signs of myocardial damage during acute myocardial &hernia. Method Coronary artery occlusion. Twenty-one male and female cats (2.3 to 3.6 kilograms) were anesthetized with sodium pentobarbital (30 mg. per kilogram) given intravenously. The trachea was cannulated and positive-pressure respiration was instituted with a Harvard respirator. Catheters were placed within the right external jugular vein and left common carotid artery and positioned for the recording of central venous pressure (CVP) and mean arterial blood pressure (MABP), respectively. Intravascular pressures were recorded continuously using appropriate Statham P-23 pressure transducers coupled to a Beckman Type R Dynograph. Needle electrodes were placed subcutaneously to allow continuous recording of Lead III of the electrocardiogram (ECG). A midsternal thoracotomy was performed, the heart exposed, and a noncannulating electromagnetic flow probe placed around the root of the aorta. The output of the flow probe was amplified by a Statham Model 4001 flowmeter and continuously recorded on the oscillographic recorder. The left coronary artery was cleared of surrounding tissue and a 3-O silk ligature was placed under the vessel. In cats subjected to acute myocardial ischemia, the ligature was tied tightly around the left coronary artery 13 to 15 mm. from the coronary ostium. Cats were either sham-operated or subjected to five hours of myocardial ischemia (MI) following occlusion of the coronary artery. Cats subjected to MI, were given either dexamethasone sodium phosphate (Decadron, Merck) 8 mg. per kilogram or an equal volume of vehicle in
July,
1975,
Vol.
90, No.
1, pp.
50-55
Effects
the jugular vein catheter. Dexamethasone was administered slowly over ten minutes beginning either 30 minutes prior to, or 60 minutes following, occlusion of the coronary artery. Shamoperated cats were given dexamethasone 30 minutes prior to the start of the experiment.
of dexamethasone on m,vwordial
Hemodynamic
Effects ligation
of Coronary
cells
Artery
Sampling and homogenization of cardiac tissue. Samples of arterial blood (4 ml.) were
withdrawn from the carotid catheter just prior to occlusion and at one, two, four, and five hours after occlusion. Blood samples were drawn from sham-operated cats at the same time. Blood loss was replaced with an equal volume of KrebsHenseleit solution warmed to 37” C. Blood was collected in polyethylene tubes containing two drops of sodium heparin, 1,000 units per milliliter (Upjohn, beef lung) and centrifuged at 2,400 x g and 4” C. for 15 minutes. The plasma was decanted and treated as described below. At five hours, the hearts were excised, rinsed in 0.9 per cent NaCl solution at 4” C., rapidly weighed, and placed in cold 0.25 M sucrose. The heart was divided into ischemic and normal left ventricle by inspection of the coronary vessels, endocardium, and epicardium. Thus, tissue supplied by arterial branches distal to the ligature appeared as a cyanotic area having patchy subendocardial hemorrhagic regions. Ischemic or nonischemic tissue was homogenized in 0.25 M sucrose (l:lO, w:v) containing 1 mM ethylenediaminetetraacetic acid and 0.1 mM mercaptoethanol for the determination of myocardial creatine phosphokinase (CPK) activity.” The tissue preparations for the CPK determinations were treated according to the method of Kjekshus and Sobel.7 Additional ischemic or adjacent normal ventricular tissue was minced and homogenized in 0.25 M sucrose (l:lO, w:v) for subsequent determination of lysosomal enzymes. Each sample of myocardium was homogenized twice for 15 seconds using a Virtis homogenizer at a speed setting of 9.0. The homogenates were centrifuged at 800 x g for 10 minutes. The supernatants were centrifuged at 36,000 x g for 30 minutes at 4O C. Supernatants were assayed for /?-glucuronidase, and cathepsin D activities in the presence and absence of Triton X-100. Cardiac homogenates from sham-operated cats were derived from left ventricular tissue anatomically equivalent to the ischemic and normal areas present in ischemic hearts. Biochemical determinations. Aliquots of the
American Heart Journal
0
1
2
lime
3
4
5
(hours)
Fig. 1. Hemodynamic effects of coronary artery ligation in cats receiving the vehicle for dexamethasone 30 minutes prior to ligation. Ligation of the left coronary artery 13 to 16 mm. from the coronaql ostium produced significant decreases in the heart rate, mean arterial blood pressure, and aortic blood flow. Values are means k S.E.M. for four cats.
plasma and supernatants of cardiac homogenates were analyzed for activities of the lysosomal protease, cathepsin D, and of another lysosomal hydrolase, P-glucuronidase, using the method of Anson* and Talalay, Fishman, and HugginsQ respectively. The protein concentration of plasma and cardiac tissue supernatants was determined using the biuret method.“’ Cathepsin D activity is expressed as milliequivalents of tyrosine x lo-” released from bovine hemoglobin per milligram of protein per hour at 37” C. Plasma and cardiac CPK activities were determined according to the method of Rosalki.” CPK activity is expressed as international units per milligram of protein. One international unit of CPK is that activity which transfers 1.0 pmole of phosphate from phosphocreatine to ADP per minute at pH 7.4 at 30” C. Results
Fig. 1 illustrates the heart rate, mean arterial blood pressure, and aortic blood flow just prior to, and for five hours following coronary ligation in untreated cats (i.e., cats receiving the vehicle for dexamethasone). Ligation of the coronary artery
51
Spath and Lefer
-Ml+V.hi.l.
TIME (hours)
2. Effect of dexamethasone (Dexa) or its vehicle upon the elevation of the S-T-segment in cats subjected to acute myocardial &hernia (MI) by occlusion of the left coronary artery. All values are means f S.E.M. for four to six cats in each group. Sham-operated cats given Dexa showed an absence of S-T-segment elevation over the entire five-hour experimental period. Occlusion of the left coronary artery initially produced similar elevations of the S-T-segment in all three groups of occluded cats. The S-T-segment of vehicletreated cats (MI + vehicle) remained elevated for the duration of the experiment. However, S-T-segment elevation declined toward control values five hours after occlusion in MI cats posttreated with Dexa (MI + Dexa, post). mV = millivolts. Fig.
produced moderate but statistically significant (p < 0.02) decreases in heart rate, mean arterial blood pressure, and aortic flow within one hour. Furthermore, the hemodynamic response of ligated cats receiving dexamethasone did not differ from that of the animals given vehicle. In contrast to cats experiencing coronary artery ligation, sham-operated cats maintained stable normal values for heart rate, blood pressure, and aortic flow over the five-hour experimental period. Furthermore, the directly measured central venous pressures and the calculated total peripheral resistances of untreated cats were similar to dexamethasone-treated cats throughout the postocclusion period. Thus, we do not find any overt hemodynamic action of dexamethasone in our experiments with regard to systemic vasodilator or positive inotropic effects. In four cats, ventricular fibrillation occurred spontaneously 20 to 30 minutes after coronary artery ligation. Cats experiencing a single episode of ventricular fibrillation were rapidly converted to sinus rhythm using a Medtronic Internal-
52
External defibrillator (Minneapolis, Minn.). For the remainder of the experimental period, these cats displayed mean hemodynamic parameters and loss of myocardial enzymes which were similar to the majority of cats without ventricular fibrillation. Cats in which the ventricle fibrillated a second time were discarded. The S-T-segment elevation recorded in dexamethasone-treated and untreated cats is shown in Fig. 2. Sham-operated cats given dexamethasone exhibited no significant change in S-T-segment elevation during five hours of observation. In contrast, marked elevation of the S-T-segment occurred at 20 to 60 minutes in all groups of cats subjected to coronary artery occlusion. Moreover, S-T-segment remained elevated for the entire experimental period in untreated cats and in cats pretreated with dexamethasone. However, posttreatment of ischemic cats with dexamethasone was associated with a significant reduction in the S-T-segment five hours after coronary artery occlusion. Concomitant with the observed electrocardiographic changes, increases in plasma CPK activities occurred in all experimental groups (Fig. 3). A significant elevation of plasma CPK activity occurred as early as two hours following coronary artery occlusion in untreated cats. Plasma CPK activity continued to increase, reaching a value of eight times the initial plasma CPK activity five hours after coronary artery occlusion. In contrast, sham-operated cats exhibited only a moderate increase in plasma CPK activity after five hours. Moreover, both pre- and posttreatment of cats with dexamethasone limited the increase in plasma CPK activity so that five hours after coronary artery occlusion, plasma CPK activity was not significantly different from sham-operated control animals. Although plasma CPK activities were altered by MI, the plasma activities of /3-glucuronidase and cathepsin D remained unchanged for the fivehour experimental period. The absence of increased plasma lysosomal hydrolase activities following myocardial ischemia correlated well with the relatively well maintained mean arterial blood pressure and aortic flow in coronary-ligated cats (Fig. 1). Adequate tissue perfusion in peripheral organs prevented loss of lysosomal hydrolases from noncardiac tissues. In contrast to the plasma activities of the lysosomal hydrolases, /3-glucuronidase and ca-
July, 1975, Vol. 90, No. 1
Elpects
thepsin D, the myocardial activities of these enzymes were affected by coronary artery ligation. Fig. 4 summarizes the myocardial cathepsin D activities of normal and ischemic myocardial tissue obtained from dexamethasone-treated and untreated cats. In cats subjected to sham MI (i.e., coronary artery isolated but not ligated) normal tissue and tissue which would have become ischemic if the coronary artery had been ligated, had very similar activities of the lysosomal protease, cathepsin D, (i.e., about 20 units per milligram of protein). Futhermore, nonischemic myocardial tissue of hearts subjected to coronary ligation also showed a cathepsin D activity approximating 20 units per milligram of protein. However, ischemic myocardial tissue of cats given the steroid vehicle exhibited a 40 per cent reduction in cathepsin D activity compared to the activity of adjacent normal tissue or to left ventricular tissue excised from sham-operated cats. In contrast, ischemic myocardial tissue of cats given dexamethasone one hour after coronary artery ligation exhibited a cathepsin D activity similar to that of normal tissue, 23.9 + 2.1 units vs. 20.7 rtr 1.4 units, respectively. However, pretreatment with dexamethasone appeared less effective in preventing the loss of myocardial cathepsin D. Thus, ischemic tissue of pretreated cats had 16 per cent less protease activity when compared to adjacent, normal tissue (p< 0.02). The alterations in myocardial CPK activities (Fig. 5) were very similar to those of cardiac cathepsin D and reflected the changes found in plasma CPK activities in the four experimental groups. Thus, the CPK activity of ischemic tissue of untreated MI cats was 40 per cent lower than the normal ventricular tissue of these hearts. Furthermore, posttreatment of MI cats with dexamethasone significantly prevented the loss of enzyme activity from the ischemic myocardium. However, pretreatment with dexamethasone was significantly less effective than posttreatment in preventing loss of CPK activity from the ischemic portion of the myocardium. Ischemic tissue of pretreated cats had 30 per cent less enzyme activity than adjacent normal tissue, a level of enzyme activity not significantly different from that observed in ischemic tissue of untreated cats. Assay of the cardiac activities of the lysosomal hydrolase /3-glucuronidase in normal and ischemic myocardium presented a similar pattern
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Fig. 3. Effect of dexamethasone or its vehicle upon plasma CPK activity before and after induction of acute myocardial &hernia (MI). All values are means rt S.E.M. expressed in International Units (IU) X lo-’ per milligram of protein. Plasma CPK activity increased eightfold in cats given vehicle prior to occlusion. A significant increase in plasma CPK occurred as early as two hours after occlusion. Treatment of cats with dexamethasone 30 minutes before or 60 minutes after occlusion prevented much of the increase in plasma CPK activity following MI.
to that observed for cardiac cathepsin D and CPK activities. Discussion
Ligation of the coronary artery produced moderate decreases in heart rate, mean arterial blood pressure, and aortic flow (Fig. 1). However, pharmacologic doses of dexamethasone given either before or after coronary artery ligation did not alter this hemodynamic pattern from that of cats given only the vehicle for dexamethasone (i.e., sterile water containing the steroid preservatives). These data indicate that dexamethasone does not alter the hemodynamic response to &hernia at least within the first five hours following coronary artery occlusion. Thus, we do not find any overt vasodilating or positive inotropic effect of dexamethasone in these experiments. This lack of a hemodynamic effect is in agreement with earlier findings’.” in dogs and in catsI wherein pharmacologic doses of dexameth-
53
Spath
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Myocardial
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Fig. 4. Effect of pre- or posttreatment with dexamethasone upon the cathepsin D activity of normal or ischemic myocardium. In cats given vehicle, myocardial ischemia resulted in 40 per cent decrease in the cathepsin D activity of ischemic ventricle relative to adjacent, normal myocardium. Pretreatment with dexamethasone reduced the loss of cathepsin D activity to 16 per cent within ischemic myocardium. Posttreatment with glucocorticoid prevented the loss of lysosomal hydrolases in acute myocardial &hernia. Values are means f S.E.M. for the number of samples shown on each bar.
Fig. 5. Effect of pre- or posttreatment with dexamethasone upon the myocardial CPK activity of normal or ischemic myocardium. All values are means + S.E.M. Numbers at the bottom of each bar indicate the number of myocardial samples studied. Ischemic myocardial tissue obtained from vehicle-treated cats (MI + vehicle) shows a 40 per cent decrease when compared with adjacent normal left ventricle. Posttreatment of cats with dexamethasone prevented the decrease in the CPK activity of ischemic heart tissue.
asone and other glucocorticoids failed to exhibit significant vascular or inotropic effects. The similarity of the hemodynamic response of ligated cats was also reflected in the early electrocardiographic changes evident in these cats. Marked elevation of the S-T-segment occurred at 20 to 60 minutes in all groups of cats subjected to coronary artery ligation indicating comparable degrees of initial myocardial injury.13 Although cats given the vehicle for dexamethasone or pretreated with the glucocorticoid maintained similarly elevated S-T-segment voltage after five hours, posttreatment of cats with dexamethasone was associated with a reduction in the S-Tsegment elevation at that time. Thus, administration of dexamethasone one hour after coronary artery ligation had a beneficial effect as assessed by the reduction of the S-T-segment elevation in the early phase of acute myocardial ischemia. The ineffectiveness of pretreatment with dexamethasone in reducing the S-T-segment elevation may be related to the plasma and tissue levels of the glucocorticoid at the time when ischemic myocardial cells began to undergo irreversible damage (i.e., about two hours after occlusion).”
Thus, pretreatment with dexamethasone results in degradation of the steroid so that inadequate concentrations are present within the ischemic portion of the myocardium at the critical twohour period. In contrast, posttreatment with dexamethasone provides a high level of glucocorticoid two hours after occlusion, the time at which the rate of cellular damage is increasing as indicated by the steep rise in plasma CPK activity of untreated cats. Determination of myocardial CPK activity of normal and ischemic tissue indicated that the observed alterations in plasma CPK activity in MI cats actually reflected loss of myocardial CPK. These findings also suggest that posttreatment with dexamethasone was more effective than pretreatment in preventing loss of CPK activity from ischemic tissue. Similar results were obtained with regard to myocardial cathepsin D activity. Dexamethasone, given after coronary artery ligation also prevented the loss of cathepsin D activity from ischemic myocardial tissue more effectively than pretreatment with the same dose of dexamethasone. The 40 per cent loss of myocardial lysosomal
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July, 1975, Vol. 90, No. 1
Effects of dexamethasone
hydrolase activity within ischemic myocardium agrees closely with the findings of Ricciutti2,15 in dogs, not only in quantity but in rate of loss. Thus, Ricciutti measured comparable loss of lysosomal hydrolase activity within four hours after coronary artery occlusion. Our results indicate that the loss of myocardial lysosomal hydrolase activity within ischemic myocardium can be significantly reduced by administration of pharmacologic doses of dexamethasone. Furthermore, these results are consistent with the hypothesis that dexamethasone stabilizes myocardial cellular or subcellular membranes within the ischemic myocardium or at the border of the evolving infarct. The membrane-stabilizing action of the glucocorticoid suggests a mechanism for the infarct-reducing effect of other glucocorticoids reported previously.‘, .i
Dexamethasone exerted no significant hemodynamic effect in sham-operated cats or in cats subjected to acute myocardial &hernia. However, the glucocorticoid did normalize elevated S-T-segments toward pre-ischemic values, and prevented much of the increase in plasma CPK activity following coronary artery ligation. Moreover, dexamethasone prevented loss of CPK activity and restricted the loss of lysosomal hydrolase within ischemic myocardial tissue. These data indicate that lysosomal disruption is an early consequence of myocardial ischemia and that treatment with dexamethasone prevents the loss of myocardial lysosomal and cellular enzymes as reflected in normalization of the ECG and plasma CPK activity of ischemic cats. In this way, dexamethasone may act to retard the spread of the developing infarct within the ischemic myocardium. The authors wish to acknowledge assistance of Mrs. Ann Gibb and Mr.
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the course of this investigation. We are also grateful for the generous gift of dexamethasone from Dr. Ingeborg Schultze of the Merck Institute of Therapeutic Research, West Point, Pa. REFERENCES 1.
2. 3.
4.
5.
6.
7.
Summary
on m,wcardial
8.
9.
10.
11.
12.
13.
14. 15.
Libby, P., Maroko, P. R., Bloor, C. M., Sobel, B. E., and Braunwald, E.: Reduction of experimental myocardial infarct size by corticosteroid administration, J. Clin. Invest. 52:599, 1973. Ricciutti, M. A.: Lysosome and myoc,ardial cellular injury, Am. J. Cardiol. 30:498, 1972. Spath, J. A., Jr., Lane, D. L., and Lefer, A. M.: Protective action of methylprednisolone on the myocardium during experimental myocardial ischemia in the> cat, Circ. Res. 35:44, 1974. Spath, J. A., Jr., Gorczynski, R. J., and Lefer, A. M.: Possible mechanisms of the beneficial action of glucocorticoids in circulatory shock, Surg. Gvnecol. Obstet. 137:597, 1973. Lefer, A. M., and Martin, J.: Mechanism of the protective effect of corticosteroids in hemorrhagic shock, Am. J. Physiol. 216:314, 1969. Rosalki, S. B.: An improved procedure for creatine phosphokinase determination, J. Lab. Clin. Med. 69:696, 1967. Kjekshus, J. K., and Sobel, B. E.: Depressed myocardial creatine phosphokinase activity following experimental myocardial infarction in rabbit, Circ. Res. 27:403, 1970. Anson, M. L.: The estimation of cathepsin with hemoglobin and the partial purification of cathepsin, J. Gen. Physiol. 20:565, 1936. Talalay, P., Fishman, W. H., and Huggins, C.: Chromogenic substrates. II. Phenolphthalein glucuronic acid as substrate for the assay of glucuronidase activity, J. Biol. Chem. 166:757, 1946. Gornall, A. G., Bardowill, C. J., and David, M. M.: Determination of serum proteins by means of the biuret reaction, J. Biol. Chem. 177:751, 1949. Gorczynski, R. J., Spath, J. A., Jr., and Lefer, A. M.: Vascular responsiveness of the in situ perfused dog pancreas, Eur. J. Pharmacol. 27:68, 1974. Kadowitz, P. J., and Yard, A. C.: Circulatory effects of hydrocortisone and protection against endotoxin shock in cats, Eur. J. Pharmacol. 9:311, 1970. Shell, W. E., Lavelle, J. F., Covell, J. W., and Sobel, B. E.: Early estimation of myocardial damage in conscious dogs and patients with evolving acute myocardial infarction, J. Clin. Invest. 52:2579, 1973. Brachfeld, N.: Maintenance of cell viability, Circ. 39:(Suppl. 4), 202, 1969. Ricciutti, M. A.: Myocardial lysosome stability in the early stages of acute ischemic injury, Am. d. Cardiol. 30:492, 1972.
the expert technical Robert Stickley during
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