Effects of acetylsalicylic acid, dipyridamole, and hydrocortisone on epinephrine-induced myocardial injury in dogs Sompong Kraikitpanitch, M.D. Carl C. Haygood Donald J. Baxter Aniece A. Yunice, Ph.D. Robert D. Lindeman, M.D. Oklahoma City, Okla.
Myocardial calcium and sodium concentrations increase and potassium and magnesium concentrations decrease in damaged or infarcted myocardium when compared to normal myocardium. ~-8 Even more striking is the increase in radiocalcium uptake, most notably into the mitochondria, in acutely damaged myocardium. T M These increases in myocardial calcium concentrations and radiocalcium uptakes into myocardium, more specifically into the mitochondrial fraction, appear to provide accurate indices of irreversible myocardial injury which might be used to quantify the cardioprotective effects of various agents or maneuvers advocated as therapy in acute myocardial infarction. Difficulty was encountered in inducing reproducible myocardial infarction in dogs by surgical ligation of coronary arteries. 7 8 Infusions of sympathetic catecholamines (isoproterenol, epinephrine, and norepinephrine) have been reported to produce diffuse myocardial necrosis in small animals. ~-~~ A diffuse and reproducible myocardial necrosis has been produced in dogs by infusion of large amounts of epinephrine over a 6 hour period. After establishing a suitable model for producing and objectively quantifying myocardial damage, the cardioprotective effects of three agents previously reported to have beneficial effects in acute myocardial infarction, From the Medical Service, Oklahoma City Veterans Administration Hospital, and Departments of Medicine and Physiology, University of Oklahoma College of Medicine, Oklahoma City, Okla. Received for publication Aug. 26, 1975. Reprint requests: Dr. Sompong Kraikitpanitch, Veterans Administration Hospital, 921 N.E. 13th St., Oklahoma City, Okla. 73104.
November, 1976, Vol. 92, No. 5, pp. 615-622
namely, acetylsalicylic acid (ASA), ~1 dipyridamole (Persantin), ~1 and hydrocortisone, 22-~4 were tested.
Methods Mongrel dogs weighing 17 to 25 kilograms were anesthetized with pentobarbital (25 mg. per kilogram) and were given small supplements as necessary to maintain anesthesia. An endotracheal tube was inserted and the animals were ventilated with a Harvard respirator. A femoral vein was cannulated for infusions and the femoral artery for blood pressure measurements and blood collections. Electrocardiograms were monitored and recorded hourly with the arterial blood pressures. The first series of studies was designed to compare the effects of epinephrine and calcium chloride against isotonic sodium chloride (control) infusions. For these studies, three groups of animals were utilized. Control group. Isotonic (0.9 per cent) sodium chloride was infused into each of the four animals at the rate of 2 c.c. per minute for 6 hours. Epinephrine group. Epinephrine (4/tg per kilogram per minute) was infused into each of seven animals in isotonic saline at the rate of 2 c.c. per minute for 6 hours. Calcium group. Calcium chloride (200 mg. per kilogram per 6 hours) was infused into each of the four animals in isotonic saline at the rate of 2 c.c. per minute for 6 hours. The second series of studies was designed to compare the protective effects of three agents (acetylsalicylic acid, dipyridamole, or hydrocorti-
American Heart Journal
615
Kraikitpanitch et al.
Table I. Myocardial concentrations (mg./lO0 Gin. fat-free dry weight) of sodium, potassium, calcium, magnesium, zinc, and copper, and of serum calcium and SGOT concentrations in animals receiving epinephrine or calcium chloride infusions compared to saline control infusions (all values, mean _+ S.E.M.)
Control No. of a n i m a l s 4 Sodium 408 +- 33 Potassium 1,320 • 68 Calcium 5.0 _+ 0.9 Magnesium 78 • 4 Zinc 6.1 • 0.1 Copper 1.6 • 0.1 Serum calcium (mEq./L.) 0 hr. 4.5 • 0.2 6 hr. 4.3 • 0.2 SGOT, 0 hr. 25 • 5 Karman activity 20 • 2 units, 6 hr.
Epinephrine
I Calcium chloride
7 4 646 • 42** 454 • 60 1,071 • 78* 1,326 + 14 12.0 • 1.1"** 7.9 • 0.9 69 • 5 77 _+ 2 5.4 • 0.5 6.2 • 0.2 1,4 • 0.3 1.8 + 0.4 4.7 • 0.2 0.2 18 + 2 202 • 30***
4.3 •
4.7 6.6 17 16
+ • • •
0.2 0.5** 3 1
*p < 0.05. **p < 0.01. *** p < 0.001.
sone) in prevention of epinephrine-induced myocardial injury. Acetylsalicylic acid group. A S A (600 mg.) was given orally" twice daily for 4 days to each of six animals prior to the epinephrine infusion described above. Dipyridamole group. Dipyridamole (50 mg.) was given orally twice daily for 4 days to each of six animals prior to the epinephrine infusion. Hydrocortisone group. Hydrocortisone (50 rag. per kilogram) was infused simultaneously with the epinephrine over the 6 hour period of infusion. All animals received 200 /~Ci of radiocalcium just prior to initiation of the epinephrine or calcium infusion. A 10 c.c. blood sample was drawn initially and at hourly intervals after starting the infusion for serum glutamic oxalacetic transaminase (SGOT), creatine phosphokinase (CPK), calcium, and blood gas determinations. After completion of the infusion, all dogs were killed with Anectine and Nembutal. The hearts were rapidly removed, placed in cellophane containers, and immersed in ice. Three samples of left ventricle were removed consistently from the same area and used immediately for the radiocalcium uptake and histology studies or stored
616
frozen for the cation concentration studies. The first myocardial sample was processed for light microscopic evaluation with hematoxylin and eosin stains. The second myocardial sample was utilized to determine concentrations of sodium, potassium, calcium, magnesium, zinc, and copper with techniques previously described. 7. 8 Samples (1 to 2 Gm.) were weighed wet, dried at 125 ~ C., and reweighed. The dried tissue was digested with 10 ml. of a mixture of nitric, sulfuric, and perchloric acid-strontium solution. With each run of samples, a blank acid mixture was treated in a similar fashion and analyzed. Cation concentrations of this blank then were subtracted from the corresponding metals of each sample and expressed in milligrams per 100 Gm. of fat-free dry weight. An aliquot of this solution was utilized to quantify zinc and copper concentrations on the Perkin Elmer 303 atomic absorption spectrophotometer, with the use of the three-slot Boling burner head with acetylene as the fuel and compressed air as the oxidizer. Aliquots of the original solution were further diluted to measure calcium, magnesium, sodium, and potassium concentrations. After linearity was established with standard solutions prepared in the same diluent, the concentrations of all four divalent cations were recorded on the direct concentration readout attachment. Sodium and potassium concentrations were quantified with a flame photometer {Instrumentation Laboratories). The third myocardial sample was used for radiocalcium uptake and differential ultracentrifugation studies by means of techniques previously described. 7 Myocardium was homogenized in 0.25M sucrose and filtered through a 4 by 4 inch gauze pad to remove muscle fibers. The filtrates were ultracentrifuged in a Beckman L-4 ultracentrifuge for 10 minutes at 700 g and the pellet which contained the nuclear fraction was isolated and weighed. The remaining homogenate was centrifuged for 60 minutes at 5,000 g. This pellet contained the mitochondrial fraction, a mixture of mitochondria and lysosomes. The remaining homogenate again was centrifuged for 60 minutes at 55,000 g and the pellet which contained the microsomal fraction, a mixture of ribosomes and sarcoplasmic reticulum, was separated. Finally, the remaining homogenate was spun for 17 hours at 80,000 g. This pellet contained soluble protein {DNA, RNA, immunoglobulins, albumin, en-
November, 1976, Vol. 92, No. 5
Epinephrine-induced myocardial injury
zymes) not attached to sarcoplasmic reticulum. The remaining supernatant was monitored in the analytic ultracentrifuge and found to be free of protein. Electron-microscopic appearances of pellets collected from infarct, peri-infarct, and normal heart have been monitored and reported previously. ~ Means and standard errors of the means were determined with standard statistical techniques. Student's paired and unpaired t tests were ultilized to test the statistical significance of observed differences. Results
The mean (_+ S.E.M.) concentrations of sodium. potassium, calcium, and magnesium per 100 Gm. of fat-free dry weight of myocardium, and serum calcium and SGOT concentrations for the control, calcium chloride, and epinephrine infusion groups of animals are tabulated in Table I. Myocardial sodium and calcium concentrations increased (p < 0.01) and potassium and magnesium concentrations decreased (p < 0.01) when the group receiving epinephrine infusions was compared against the control group. Serum SGOT concentrations also were increased significantly (p < 0.01}. No significant differences in any myocardial cation concentration was observed in the calcium chloride infusion animals compared to control animals. T he serum calcium concentrations were significantly increased (p < 0.01) over those observed in the control animals. 45Ca uptakes into intact myocardium and each of its subcellular components {nuclear, mitochondrial, microsomal, soluble protein} of epinephrine, calcium chloride, and control groups are shown in Table II. Significant increases (p < 0.05) in radiocalcium uptakes were found in intact myocardium and in all subcellular fractions in the epinephrine-infused animals compared against the control animals. T he 45Ca uptakes into intact myocardium and the subcellular components tended to be lower in the calcium chloride-infused animals but the decrease was significant (p < 0.01) only in the microsomal fraction. A comparison of the morphologic evidence of myocardial damage in the first three groups is shown in Table III. Evidence of myocardial damage was graded without prior knowledge of treatment by one observer (D. J. B.) as normal,
American Heart Journal
Table ,,. Radiocalcium ('~Ca) uptakes (no. of
counts per minute per gram of dried tissue or fraction) into intact myocardium and each of its subcellular components {nuclear, mitochondrial, microsomal, soluble protein) in animals receiving epinephrine or calcium chloride infusions compared to saline control infusions (all values, mean _+ S.E.M.)
I No. of animals Nuclear Mitochondrial Microsomal Soluble protein Intact myocardium
b
Control
Epinephrine
4 317 827 751 429 334
+_ + + _~ ~
a,c/u
chloride
7 40 76 86 136 57
2,148 4,957 3,158 2.085 2.860
4
_+ 463** _+ 324*** _+ 568** _+ 497** _~ 883*
243 599 376 223 242
_+ + + z z
25 194 44** 49 16
~p
Myocardial calcium and sodium concentrations increase and potassium and magnesium concentrations decrease in damaged or infarcted myocardium when compared to normal myocardium. ~-8 Even more striking is the increase in radiocalcium uptake, most notably into the mitochondria, in acutely damaged myocardium. T M These increases in myocardial calcium concentrations and radiocalcium uptakes into myocardium, more specifically into the mitochondrial fraction, appear to provide accurate indices of irreversible myocardial injury which might be used to quantify the cardioprotective effects of various agents or maneuvers advocated as therapy in acute myocardial infarction. Difficulty was encountered in inducing reproducible myocardial infarction in dogs by surgical ligation of coronary arteries. 7 8 Infusions of sympathetic catecholamines (isoproterenol, epinephrine, and norepinephrine) have been reported to produce diffuse myocardial necrosis in small animals. ~-~~ A diffuse and reproducible myocardial necrosis has been produced in dogs by infusion of large amounts of epinephrine over a 6 hour period. After establishing a suitable model for producing and objectively quantifying myocardial damage, the cardioprotective effects of three agents previously reported to have beneficial effects in acute myocardial infarction, From the Medical Service, Oklahoma City Veterans Administration Hospital, and Departments of Medicine and Physiology, University of Oklahoma College of Medicine, Oklahoma City, Okla. Received for publication Aug. 26, 1975. Reprint requests: Dr. Sompong Kraikitpanitch, Veterans Administration Hospital, 921 N.E. 13th St., Oklahoma City, Okla. 73104.
November, 1976, Vol. 92, No. 5, pp. 615-622
namely, acetylsalicylic acid (ASA), ~1 dipyridamole (Persantin), ~1 and hydrocortisone, 22-~4 were tested.
Methods Mongrel dogs weighing 17 to 25 kilograms were anesthetized with pentobarbital (25 mg. per kilogram) and were given small supplements as necessary to maintain anesthesia. An endotracheal tube was inserted and the animals were ventilated with a Harvard respirator. A femoral vein was cannulated for infusions and the femoral artery for blood pressure measurements and blood collections. Electrocardiograms were monitored and recorded hourly with the arterial blood pressures. The first series of studies was designed to compare the effects of epinephrine and calcium chloride against isotonic sodium chloride (control) infusions. For these studies, three groups of animals were utilized. Control group. Isotonic (0.9 per cent) sodium chloride was infused into each of the four animals at the rate of 2 c.c. per minute for 6 hours. Epinephrine group. Epinephrine (4/tg per kilogram per minute) was infused into each of seven animals in isotonic saline at the rate of 2 c.c. per minute for 6 hours. Calcium group. Calcium chloride (200 mg. per kilogram per 6 hours) was infused into each of the four animals in isotonic saline at the rate of 2 c.c. per minute for 6 hours. The second series of studies was designed to compare the protective effects of three agents (acetylsalicylic acid, dipyridamole, or hydrocorti-
American Heart Journal
615
Kraikitpanitch et al.
Table I. Myocardial concentrations (mg./lO0 Gin. fat-free dry weight) of sodium, potassium, calcium, magnesium, zinc, and copper, and of serum calcium and SGOT concentrations in animals receiving epinephrine or calcium chloride infusions compared to saline control infusions (all values, mean _+ S.E.M.)
Control No. of a n i m a l s 4 Sodium 408 +- 33 Potassium 1,320 • 68 Calcium 5.0 _+ 0.9 Magnesium 78 • 4 Zinc 6.1 • 0.1 Copper 1.6 • 0.1 Serum calcium (mEq./L.) 0 hr. 4.5 • 0.2 6 hr. 4.3 • 0.2 SGOT, 0 hr. 25 • 5 Karman activity 20 • 2 units, 6 hr.
Epinephrine
I Calcium chloride
7 4 646 • 42** 454 • 60 1,071 • 78* 1,326 + 14 12.0 • 1.1"** 7.9 • 0.9 69 • 5 77 _+ 2 5.4 • 0.5 6.2 • 0.2 1,4 • 0.3 1.8 + 0.4 4.7 • 0.2 0.2 18 + 2 202 • 30***
4.3 •
4.7 6.6 17 16
+ • • •
0.2 0.5** 3 1
*p < 0.05. **p < 0.01. *** p < 0.001.
sone) in prevention of epinephrine-induced myocardial injury. Acetylsalicylic acid group. A S A (600 mg.) was given orally" twice daily for 4 days to each of six animals prior to the epinephrine infusion described above. Dipyridamole group. Dipyridamole (50 mg.) was given orally twice daily for 4 days to each of six animals prior to the epinephrine infusion. Hydrocortisone group. Hydrocortisone (50 rag. per kilogram) was infused simultaneously with the epinephrine over the 6 hour period of infusion. All animals received 200 /~Ci of radiocalcium just prior to initiation of the epinephrine or calcium infusion. A 10 c.c. blood sample was drawn initially and at hourly intervals after starting the infusion for serum glutamic oxalacetic transaminase (SGOT), creatine phosphokinase (CPK), calcium, and blood gas determinations. After completion of the infusion, all dogs were killed with Anectine and Nembutal. The hearts were rapidly removed, placed in cellophane containers, and immersed in ice. Three samples of left ventricle were removed consistently from the same area and used immediately for the radiocalcium uptake and histology studies or stored
616
frozen for the cation concentration studies. The first myocardial sample was processed for light microscopic evaluation with hematoxylin and eosin stains. The second myocardial sample was utilized to determine concentrations of sodium, potassium, calcium, magnesium, zinc, and copper with techniques previously described. 7. 8 Samples (1 to 2 Gm.) were weighed wet, dried at 125 ~ C., and reweighed. The dried tissue was digested with 10 ml. of a mixture of nitric, sulfuric, and perchloric acid-strontium solution. With each run of samples, a blank acid mixture was treated in a similar fashion and analyzed. Cation concentrations of this blank then were subtracted from the corresponding metals of each sample and expressed in milligrams per 100 Gm. of fat-free dry weight. An aliquot of this solution was utilized to quantify zinc and copper concentrations on the Perkin Elmer 303 atomic absorption spectrophotometer, with the use of the three-slot Boling burner head with acetylene as the fuel and compressed air as the oxidizer. Aliquots of the original solution were further diluted to measure calcium, magnesium, sodium, and potassium concentrations. After linearity was established with standard solutions prepared in the same diluent, the concentrations of all four divalent cations were recorded on the direct concentration readout attachment. Sodium and potassium concentrations were quantified with a flame photometer {Instrumentation Laboratories). The third myocardial sample was used for radiocalcium uptake and differential ultracentrifugation studies by means of techniques previously described. 7 Myocardium was homogenized in 0.25M sucrose and filtered through a 4 by 4 inch gauze pad to remove muscle fibers. The filtrates were ultracentrifuged in a Beckman L-4 ultracentrifuge for 10 minutes at 700 g and the pellet which contained the nuclear fraction was isolated and weighed. The remaining homogenate was centrifuged for 60 minutes at 5,000 g. This pellet contained the mitochondrial fraction, a mixture of mitochondria and lysosomes. The remaining homogenate again was centrifuged for 60 minutes at 55,000 g and the pellet which contained the microsomal fraction, a mixture of ribosomes and sarcoplasmic reticulum, was separated. Finally, the remaining homogenate was spun for 17 hours at 80,000 g. This pellet contained soluble protein {DNA, RNA, immunoglobulins, albumin, en-
November, 1976, Vol. 92, No. 5
Epinephrine-induced myocardial injury
zymes) not attached to sarcoplasmic reticulum. The remaining supernatant was monitored in the analytic ultracentrifuge and found to be free of protein. Electron-microscopic appearances of pellets collected from infarct, peri-infarct, and normal heart have been monitored and reported previously. ~ Means and standard errors of the means were determined with standard statistical techniques. Student's paired and unpaired t tests were ultilized to test the statistical significance of observed differences. Results
The mean (_+ S.E.M.) concentrations of sodium. potassium, calcium, and magnesium per 100 Gm. of fat-free dry weight of myocardium, and serum calcium and SGOT concentrations for the control, calcium chloride, and epinephrine infusion groups of animals are tabulated in Table I. Myocardial sodium and calcium concentrations increased (p < 0.01) and potassium and magnesium concentrations decreased (p < 0.01) when the group receiving epinephrine infusions was compared against the control group. Serum SGOT concentrations also were increased significantly (p < 0.01}. No significant differences in any myocardial cation concentration was observed in the calcium chloride infusion animals compared to control animals. T he serum calcium concentrations were significantly increased (p < 0.01) over those observed in the control animals. 45Ca uptakes into intact myocardium and each of its subcellular components {nuclear, mitochondrial, microsomal, soluble protein} of epinephrine, calcium chloride, and control groups are shown in Table II. Significant increases (p < 0.05) in radiocalcium uptakes were found in intact myocardium and in all subcellular fractions in the epinephrine-infused animals compared against the control animals. T he 45Ca uptakes into intact myocardium and the subcellular components tended to be lower in the calcium chloride-infused animals but the decrease was significant (p < 0.01) only in the microsomal fraction. A comparison of the morphologic evidence of myocardial damage in the first three groups is shown in Table III. Evidence of myocardial damage was graded without prior knowledge of treatment by one observer (D. J. B.) as normal,
American Heart Journal
Table ,,. Radiocalcium ('~Ca) uptakes (no. of
counts per minute per gram of dried tissue or fraction) into intact myocardium and each of its subcellular components {nuclear, mitochondrial, microsomal, soluble protein) in animals receiving epinephrine or calcium chloride infusions compared to saline control infusions (all values, mean _+ S.E.M.)
I No. of animals Nuclear Mitochondrial Microsomal Soluble protein Intact myocardium
b
Control
Epinephrine
4 317 827 751 429 334
+_ + + _~ ~
a,c/u
chloride
7 40 76 86 136 57
2,148 4,957 3,158 2.085 2.860
4
_+ 463** _+ 324*** _+ 568** _+ 497** _~ 883*
243 599 376 223 242
_+ + + z z
25 194 44** 49 16
~p
