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Tritiated digoxin. XX. Tissue distribution in experimental myocardial infarction
Tritiated digoxin. XX. Tissue distribution experimental myoeardial infarction
in
A. J. Thompeon, Major, USAF, MC* J. Hargis, M.D. M. L. Murphy, M.D. J. E. Doherty, M.D. Brooks Air Force Base, Texas, and Little Rock, Ark.
Experimental myocardial infarction has been reported to increase myocardial sensitivity to digitalis glycosides.1-5 The controversy surrounding the possible hazards of digitalis administration in acute myocardial infarction has been the subject of a number of reports and reviews.6-” Pathologic studies demonstrate islands of viable myocardium in areas of infarction,12 and electrophysiologic studies reveal electrically unstable areas in infarcted and ischemic myocardium.13s l4 Recent evidence suggests that intoxication with digitalis glycosides may occur at lower serum concentrations in patients with atherosclerotic heart disease and myocardial infarction.15v l6 The known digitalis effect of enhanced automaticity in Purkinje fibers and focal re-excitation of ventricular muscle13 led us to investigate the concentration of tritiated digoxin in normal, ischemic, and infarcted left ventricular myocardium in an effort to more clearly define the role of digitalis in myocardial infarction. From the Department of Medicine, Division of Cardiology, University of Arkansas Medical Center, and the Veterans Administration Hospital, Little Rock, Ark. Supported in part by United States 06642-11 and the Burroughs-Wellcome Park, N. C. Received
for publication
Public Health Service Grant & Co., Inc., Research Triangle
Oct. 22, 1973.
Reprint requests to: A. J. Thompson, Major, USAF, MC, USAF/SAM/ NGI, Brooks Air Force Base, Texas 78236, or, Dr. J. E. Doherty, Director, Division of Cardiology, VA-UAMC Complex, 300 E. Roosevelt Rd., Little Rock, Ark. 72206. ‘Dr. Thompson is currently with the Clinical Sciences Division, United States Air Force School of Aerospace Medicine, Aerospace Medical Division, United States Air Force, Brooks Air Force Base, Texas.
September,
1974, Vol. 88, No. 3, pp. 319-324
Materials and methods
Eleven healthy mongrel dogs* weighing an average of 18.1 kilograms (+- 1.4 kilograms S.E.M.) were anesthetized with pentobarbital(25 mg. per kilogram of body weight). Ventilation with room air was maintained with .an endotracheal tube and respirator. The femoral artery was cannulated for continuous pressure monitoring and sampling of blood. The external jugular vein was cannulated for administration of drugs. Electrocardiographic Leads I, aV,, and the arterial pressure were displayed and recorded (50 mm. per second) on a multi-channel Electronics for Medicine recorder. Following left thoracotomy, ligation of the anterior interventricular coronary artery between the first and second perforating branches produced an anterolateral myocardial infarction. All animals developed ST-segment elevation and Qwaves characteristic of infarction. Ventricular arrhythmias developed in a number of experiments and were treated when necessary with intravenous lidocaine. Five minutes after ligation of the coronary artery, 0.6 mg. of tritiated digoxin was given intravenously. The tritiateddigoxin which was prepared by the Wilxbach hydrogen exchange method,17 was chemically and chromatographically pure.l*The specific activity of the lots used were 125 mCi per milligram and 161 mCi per milligram. Arterial blood for serum digoxin deter*The animals involved in this study were maintained and used in accordance with the Animal Welfare Act of 1970 and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences-National Research Council.
American
Heart Journal
319
Thompson
et al.
Fig. 1. Cross-sectional tissue slice through the ventricles. As differentiated by the vital dye, the normal myocardium stains dark green, the ischemic border zone is gray, and the area of normal coloration represents the infarcted zone. Tissue samples were taken from each zone to determine tritiated digoxin content and oxidative phosphorylation measurements.
mination was collected at 5,10,X, 30,45,60,90, and 120 minutes from the time of digoxin administration. Two hours following the administration of intravenous tritiated digoxin, a vital dye (20 mg. per kilogram of 10 per cent solution of alphazurine 2G) was rapidly injected into the venous catheter. The heart was quickly removed from the chest and immediately taken into a tissue preparation room where all further dissections were done. Following cross-sectional slicing of the heart, full-thickness tissue samples were taken from the normal, ischemic, and infarcted zone. Tissues were weighed and homogenized with a small amount of distilled water. The individual specimens were extracted with chloroform and passed through an alumina column. The column was eluted with 2:l chloroform-ethanol mixture. The eluate was evaporated, the counting solution was added, and radioactivity was measured with a liquid scintillation spectrometer (Packard TriCarb 21). The results were expressed as: nanograms of tritiated digoxin per milliliter for serum and nanqgrams of tritiated digoxin per gram of wet weight, for tissue. The differentiation between the zones was determined by dye distribution (Fig. 1). The polarographic oxygen electrode technique
320
was used to study mitochondrial respiration and oxidative phosphorylationlg* aoof normal and infarcted myocardium in six experiments. The mitochondrial pellet was prepared from approximately 7 Gm. of myocardium which was washed with homogenizing solution, minced, and homogenized.21 Differential centrifugation was then carried out and the mitochondrial pellet was suspended in malate or glutamate substrate. All tissue was processed at 0 to 4” C. and studies began in less than 90 minutes after death of the animal. Simultaneous determination of the normal and infarcted myocardium in the two substrates allowed maximal comparison. Mitochondrial oxygen consumption (microatoms of oxygen consumed per milligram of mitochondrial protein per minute), ADP/O ratio (micromoles of adenosinediphosphate [ADPI phosphorylated per atom of oxygen consumed), and respiratory control index (the ratio of oxygen consumption in the presence of ADP compared to that after ADP) were determined for each tissue and substrate.21 Mitochondrial protein was determined by standard methods, and ADP assays were determined spectrophotometrically. Results Serum samples taken at frequent intervals after the intravenous administration of 0.5 mg. of tritiated digoxin revealed rapid disappearance of digoxin from the serum. Within a 2-hour period after 0.5 mg. of digoxin was administered intravenously, the serum concentration fell from approximately 35 ng. per milliliter at five minutes to 2.1 ng. per milliliter (Fig. 2). It has previously been documented that this rapid fall in serum concentration primarily represents organ distribution and binding,22 and that two hours allow for equilibration of the serum and tissue compartmentally. Myocardial tissue samples taken two hours after the intravenous administration of 0.5 mg. of tritiated digoxin revealed a significant variation in digoxin concentration in and around the infarcted zone (Fig. 3). The concentration of 90 ng. of tritiated digoxin per gram of normal left ventricle and a tissue-to-serum ratio of 43:l is in close agreement with findings previously published by this laboratory.23 The infarcted tissue demonstrated a tissue-to-serum ratio of 12:1, a 3- to 4-fold decrease in tritiated digoxin content as compared to normal myocardium. The
September,
1974, Vol. 88, No. 3
‘l’ritiated
digoxin
I
SE
l.O!
0
I
I
1
I
I5
30
45
60
f
75
6
90
105
120
MINUTES FIQ. 2. Serum disappearance serum concentration (mean horizontal axis.
curve of 3H-digoxin f S.E.M.) is plotted
following the intravenous on the vertical axis, with
injection of 0.5 semilogarithmic
mg. of aH-diguxin. ecale and time
The on the
Table I Wet wei& Experiment No. 1 2 3 4 5 6 I 8 9 10 11 Mean S.E.M. &
Weight OQ..) 13.6 18.0 14.1 23.2 14.5 22.7 12.7 16.4 17.3 25.5 20.9 18.1 1.4
3Hdigoxin dose hg.)
Serum concentration (ng.lml.)
Normal LV
Ischemic LV
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 -
2.32 1.54 1.85 2.22 3.26 1.48 3.38 2.43 2.17 0.90 1.76 2.12 0.67
150.35 84.51 74.28 105.50 114.22 66.10 163.17 70.88 69.75 52.26 29.37 89.13 28.19
130.35 38.35 13.71 39.18 106.26 41.08 99.95 70.34 66.61 33.60 23.52 60.27 19.06
ischemic xone was intermediate between normal and infarct, and a myocardial tissue level of 55 ng. per gram and ratio of 26:l was present. The serum concentration of tritiated digoxin in the cardiac tissues sampled in each of the eleven animal preparation8 is listed in Table I. Tissue samples from the right ventricle, atrium, and pericardial fat were analyzed for
American Heart Journal
Znfareted LV 9.51 18.92 7.71 33.30 48.43 20.54 27.92 26.54 49.82 11.60 1.53 23.98 7.58
fng.lGm.) Pericardial RV
Atrium
fat
92.48 55.32 55.10 67.70 88.16 46.91 127.09 58.26 64.07 45.71 37.42 67.07 21.21
73.98 38.69 44.41 1.06 70.33 28.76 73.23 32.15 38.17 23.85 12.65 45.45 14.37
3.95 2.38 4.66 1.68 4.95 2.01 2.46 0.43 1.50 2.51 kO.51
digoxin content. The normal right ventricle contained less digoxin per gram of tissue than did the normal left ventricle. Atria1 tissue concentrated less digoxin per gram of tissue than did either ventricle. The pericardial fat had a digoxin concentration not significantly different from that of serum. Listed in Table II are the data evaluating the
321
Thompson et al. Table II RCR
@2 Experiment No.
3 5 6 7 10 11 Mean S.E.M. t-
Normal
Infarct
0.4791 0.3979 0.3003 0.2895 0.2890 0.2867 0.3404 0.036
0.0856 0.1649 0.0957 0.0977 0.0823 0.0867 0.1023 0.014
-
NORMAL L.V.
ISCHEMIC L v.
INFARCTED LV
/
Normal
Znfarct
Normal
3.61 3.87 4.02 3.78 4.31 4.24 3.97 0.12
2.12 2.97 2.35 3.03 2.21 2.29 2.50 0.18
2.31 2.38 2.78 2.75 2.89 2.63 2.62 0.10
Infarct
1.87 2.56 2.15 2.09 2.17 2.30 2.19 0.10
specialized equipment. Oxidative phosphorylation determination confirmed the division of tissue into normal and infarcted regions by demonstrating impaired oxygen utilization of mitochondria from infarcted myocardium. Right ventricular myotardium has been shown to concentrate less digoxin than the left ventricle per gram of tissue weight. Atria1 tissue concentrates less digoxin than either ventricle, and pericardial fat appeared to have no tissue-to-serum gradient. The studies revealed a marked disparity of tritiated digoxin distribution in the canine left ventricle after acute myocardial infarction. Homogeneous distribution of tritiated digoxin in the normal canine left ventricle has been demonstrated in previous studies.22,24After intravenous administration, myocardial concentration of tritiated digoxin peaks at one hour and is well maintained for 12 hours in the caninez6; therefore, the two-hour tissue samples should be representative of maximal myocardial concentration. Altered regional blood flow to ischemic myocardium may explain the distribution pattern of tritiated digoxin, as suggested by radioisotope studies utilizing labeled carbonized microspheres26* 27 and i31 I-iodoantipyrine.28 The ischemic myocardium may also show reduced tritiated digoxin uptake due to high extracellular potassium concentrationzs which accumulates from cellular injury and intracellular potassium loss. Normal myocardium has a tissue concentration and tissue-to-serum ratio which closely agrees with previous findings in our laboratory. The myocardial digoxin content decreases progressively in normal, ischemic, and infarcted
I..-Rl”M
PERICARDIAL FAT
Fig. 3. Myocardial concentration of 3H-digoxin. Tissue samples were taken two hours after the intravenous administration of 0.5 mg. of 3H-digoxin. Digoxin concentration in nanograms per gram of wet weight tissue (mean f S.E.M.) is on the ordinate. Concentration in the normal left ventricle is approximately 90 ng. per gram of tissue. The ischemic left ventricle and the infarcted left ventricle contained 54 and 25 ng. per gram of tissue, respectively. The normal right ventricle tritiated digoxin concentration of 64 ng. par gram of tissue is less than the normal left ventricular myocardium. Atria1 tissue contains less tritiated digoxin than either ventricle. Pericardial fat has a concentration of tritiated digoxin equivalent to serum.
metabolic activity of mitochondria from normal and infarcted myocardium in six of the animal preparations. Tissue respiration in malate substrate-as measured by oxygen consumption, respiratory control index, and ADP/O-is signiIicantly reduced in the infarcted tissue, verifying tissue hypoxia and altered mitochondrial integrity in the infarcted zones. Discussion
The normal, ischemic, and infarcted myocardial zones were easily differentiated with a vital dye. This rapid and accurate method required no
322
ADPIO
September,1974, Vol. 88, No. 3
Vitiated
tissue with a marked tritiated digoxin gradient being present between these respective zones. Disparate repolarization and the initiation of reentrant ectopic rhythms have been demonstrated in ischemic myocardium.13v l4 Canine Purkinje fibers exhibit enhanced automaticity in the presence of hypoxia and fiber stretch.30 The similar digitalis effect of enhanced automaticity of Purkinje fibers and facilitation of ectopic rhythms is potentially additive in the ischemic myocardium.31, 32Thus, a marked digoxin gradient between normal, ischemic, and infarcted myocardium may potentiate the appearance of ectopic rhythms from electrically unstable ischemic or infarcted myocardium.
6. 7. 8.
9. 10. 11. 12.
13.
Summary
The role and potential hazards of digitalis glycoside administration in acute myocardial infarction remain controversial. We investigated the concentration of tritiated digoxin in normal, ischemic, and infarcted left ventricular myocardium of the dog after ligation of the anterior interventricular coronary artery. The normal homogeneous distribution of tritiated digoxin in the normal canine left ventricle was altered following acute myocardial infarction. The ischemic and infarcted zones exhibited a marked diminution in digoxin concentration. Oxidative phosphorylation determinations confirmed tissue hypoxia in the infarcted zone. The gradient of digoxin concentration between normal, ischemic, and infarcted zones of myocardium may potentiate the development of an arrhythmia in the electrically unstable infarcted myocardium. We would like to acknowledge the technical assistance of William Lynch, Jacquelyn Gammill, B.S., M.T.(A.S.C.P.), and Joyce Sherwood, B.S., M.T.(A.S.C.P.). Gratitude is expressed to Mrs. Genevieve Lopez for typing of this manuscript. REFERENCES 1.
2.
3.
4.
5.
Travell, J., Gold, H., and Modell, W.: Effect of experimental cardiac infarction on response to digitalis, Arch. Intern. Med. 61:184, 1938. Bellet, S., Johnston, C. G., and Schecter, A.: Effect of cardiac infarction on the tolerance of dogs to digitalis, Arch. Intern. Med. W:509,1934. Hood, W. B., Jr., McCarthy, B., and Lown, B.: Myocardial infarction following coronary ligation in dogs: Hemodynamic effects of isoproterenol and acetylstrophanthidin, Circ. Res. 21:191, 1967. Morris, J. J., Taft, C. V., Whalen, R. E., and McIntosh, H. D.: Digitalis and experimental myocardial infarction, AM. HEART J. 77:342,1969. Kumar, R., Hood, W. B., Jr., Jo&on, J., Gilmour, D. P.,
American Heart Journal
14. 15.
16.
17.
18.
19.
20.
21.
22. 23.
24.
25.
26.
27.
28.
digoxin
Norman, J, C., and Abelmann, W. H.: Experimental myocar&al infarction. VI. Efficacy and toxicity of digitalis in acute and healing phase in intact conscious dogs, J. Clin. Invest. 49:356, 1970. &key, J. M.: Digitalis in acute myocardial infarction, J. A. M. A. 146:1008, 1951. Bayer, N. H.: Digitalis in acute myocardial infarction, N. Engl. J. Med. 262:536, 1965. Shrager, M. W.: Digitalis intoxication: A review and report of forty cases with emphasis on etiology. Arch. Intern. Med. 100881, 1957. Somylo, A. P.: The toxicology of digitalis, Am. J. Cardiol. 5523, 1960. Selzer, A.: The use of digitalis in acute myocardial infarction, Progr. Cardiovasc. Dis. 10518, 1968. Constant, J.: Digitalis in myocardial infarction, N. Y. J. Med. 70~650, 1970. Mallory, G. K., White, P. D., and Salcedo-Salgar, J.: The sneed of healing of myocardial infarction, AM. HEART J. 16:647, 1939. Han, J., and Moe, G. K.: Nonuniform recovery of excitability in ventricular muscle, Circ. Res. 14~44, 1964. Han, J.: The concepts of re-entrant activity responsible for ectopic rhythms, Am. J. Cardiol. 28:253, 1971. Beller, G. A., Smith, T. W., Abelmann, W. H., Haber, E., and Hood, W. B., Jr.: Digitalis intoxication: a prospective clinical study with serum level correlations, N. Engl. J. Med. 284:989, 1971. Smith, T. W.: Digitalis: Contribution of quantitative ass&y technics to the understanding of the clinical pharmacology of digitalis, Circulation 46~188, 1972. Wilzbach, K. E.: Tritium-labeling by exposure of organic compounds to tritium gas, J. Am. Chem. Sot. 79:1013, 1957. Doherty, J. E., Perkins, W. H., and Mitchell, G. K.: Tritiated digoxin studies in human subjects, Arch. Intern. Med. 108531, 1961. Chance, B., and Williams, G. R.: A simple and rapid assay of oxidative phosphorylation, Nature 175:1120, 1955. Estabrook, R. W.: Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios, Oxidation and Phosphorylation, Meth. Enzymol. 10:41, , 1967. Sobel, B., Jequier, E., Sjoerdsma, A., and Lovenberg, W.: Effect of catecholamines and adrenergic blocking agents on oxidative phosphorylation in rat heart mitochondria, Circ. Res. 19:1050, 1966. Doherty, J. E.: The clinical pharmacology of digitalis glycosides: A review, Am. J. Med. Sci. 255:382, 1968. Doherty, J. E., and Perkins, W. H.: Tissue concentration and turnover of tritiated digoxin in dogs, Am. J. Cardial. 17:47, 1966. Beller, G. A., Smith, T. W., and Hood, W. B., Jr.: Altered distribution of tritiated digoxin in the infarcted canine left ventricle, Circulation 46572, 1972. Deutscher, R. N., Harrison, D. C., and Goldman, R. H.: The relation between myocardial 3H-digoxin concentration and its hemodynamic effects, Am. J. Cardiol. 29:47, 1972. Becker, L. C., Fortuin, N. J., and Pitt, B.: Effect of ischemia and anti-angina1 drugs on the distribution of radioactive microspheres in the canine left ventricle, Circ. Res. 28:263, 1971. Portuin, N. J., Kaihara, S., Becker, L. C., and Pitt, B.: Regional myocardial blood flow in the dog studied with radioactive microspheres, Cardiovasc. Reg. S:331,1971. Griggs, D. M., Jr., and Nakamura. Y.: Effect of coronary
323
Thompson
et al.
constriction on myocardial distribution of iodoantipyrine-1311, Am. J. Phyeiol. 215:1082,1968. 29. Marcus, F. I., Kapadia, G. G., and Goldsmith, C.: The alteration of the body distribution of tritiated digoxin by acute hyperkalemia in the dog, J. Pharmacol. Exp. Ther. 165:136, 1969. 30. Han, J.: Mechanisms of ventricular arrhythmias associated with myocardial infarction, Am. J. Cardiol. 24800, 1969.
324
31. Hoffman, B. F., and Singer, D. H.: Effects of digitalis on electrical activity of cardiac fibers, Progr. Cardiovasc. Dis. 7:226, 1964. 32. Vaesalle, M., Karis, J., and Hoffman, B. F.: Toxic effects of ouabain on Purkiqje fibers and ventricular muscle fibers, Am. J. Physiol. 203:433, 1962.
September, 1974, Vol. 88, No. 3
in
A. J. Thompeon, Major, USAF, MC* J. Hargis, M.D. M. L. Murphy, M.D. J. E. Doherty, M.D. Brooks Air Force Base, Texas, and Little Rock, Ark.
Experimental myocardial infarction has been reported to increase myocardial sensitivity to digitalis glycosides.1-5 The controversy surrounding the possible hazards of digitalis administration in acute myocardial infarction has been the subject of a number of reports and reviews.6-” Pathologic studies demonstrate islands of viable myocardium in areas of infarction,12 and electrophysiologic studies reveal electrically unstable areas in infarcted and ischemic myocardium.13s l4 Recent evidence suggests that intoxication with digitalis glycosides may occur at lower serum concentrations in patients with atherosclerotic heart disease and myocardial infarction.15v l6 The known digitalis effect of enhanced automaticity in Purkinje fibers and focal re-excitation of ventricular muscle13 led us to investigate the concentration of tritiated digoxin in normal, ischemic, and infarcted left ventricular myocardium in an effort to more clearly define the role of digitalis in myocardial infarction. From the Department of Medicine, Division of Cardiology, University of Arkansas Medical Center, and the Veterans Administration Hospital, Little Rock, Ark. Supported in part by United States 06642-11 and the Burroughs-Wellcome Park, N. C. Received
for publication
Public Health Service Grant & Co., Inc., Research Triangle
Oct. 22, 1973.
Reprint requests to: A. J. Thompson, Major, USAF, MC, USAF/SAM/ NGI, Brooks Air Force Base, Texas 78236, or, Dr. J. E. Doherty, Director, Division of Cardiology, VA-UAMC Complex, 300 E. Roosevelt Rd., Little Rock, Ark. 72206. ‘Dr. Thompson is currently with the Clinical Sciences Division, United States Air Force School of Aerospace Medicine, Aerospace Medical Division, United States Air Force, Brooks Air Force Base, Texas.
September,
1974, Vol. 88, No. 3, pp. 319-324
Materials and methods
Eleven healthy mongrel dogs* weighing an average of 18.1 kilograms (+- 1.4 kilograms S.E.M.) were anesthetized with pentobarbital(25 mg. per kilogram of body weight). Ventilation with room air was maintained with .an endotracheal tube and respirator. The femoral artery was cannulated for continuous pressure monitoring and sampling of blood. The external jugular vein was cannulated for administration of drugs. Electrocardiographic Leads I, aV,, and the arterial pressure were displayed and recorded (50 mm. per second) on a multi-channel Electronics for Medicine recorder. Following left thoracotomy, ligation of the anterior interventricular coronary artery between the first and second perforating branches produced an anterolateral myocardial infarction. All animals developed ST-segment elevation and Qwaves characteristic of infarction. Ventricular arrhythmias developed in a number of experiments and were treated when necessary with intravenous lidocaine. Five minutes after ligation of the coronary artery, 0.6 mg. of tritiated digoxin was given intravenously. The tritiateddigoxin which was prepared by the Wilxbach hydrogen exchange method,17 was chemically and chromatographically pure.l*The specific activity of the lots used were 125 mCi per milligram and 161 mCi per milligram. Arterial blood for serum digoxin deter*The animals involved in this study were maintained and used in accordance with the Animal Welfare Act of 1970 and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences-National Research Council.
American
Heart Journal
319
Thompson
et al.
Fig. 1. Cross-sectional tissue slice through the ventricles. As differentiated by the vital dye, the normal myocardium stains dark green, the ischemic border zone is gray, and the area of normal coloration represents the infarcted zone. Tissue samples were taken from each zone to determine tritiated digoxin content and oxidative phosphorylation measurements.
mination was collected at 5,10,X, 30,45,60,90, and 120 minutes from the time of digoxin administration. Two hours following the administration of intravenous tritiated digoxin, a vital dye (20 mg. per kilogram of 10 per cent solution of alphazurine 2G) was rapidly injected into the venous catheter. The heart was quickly removed from the chest and immediately taken into a tissue preparation room where all further dissections were done. Following cross-sectional slicing of the heart, full-thickness tissue samples were taken from the normal, ischemic, and infarcted zone. Tissues were weighed and homogenized with a small amount of distilled water. The individual specimens were extracted with chloroform and passed through an alumina column. The column was eluted with 2:l chloroform-ethanol mixture. The eluate was evaporated, the counting solution was added, and radioactivity was measured with a liquid scintillation spectrometer (Packard TriCarb 21). The results were expressed as: nanograms of tritiated digoxin per milliliter for serum and nanqgrams of tritiated digoxin per gram of wet weight, for tissue. The differentiation between the zones was determined by dye distribution (Fig. 1). The polarographic oxygen electrode technique
320
was used to study mitochondrial respiration and oxidative phosphorylationlg* aoof normal and infarcted myocardium in six experiments. The mitochondrial pellet was prepared from approximately 7 Gm. of myocardium which was washed with homogenizing solution, minced, and homogenized.21 Differential centrifugation was then carried out and the mitochondrial pellet was suspended in malate or glutamate substrate. All tissue was processed at 0 to 4” C. and studies began in less than 90 minutes after death of the animal. Simultaneous determination of the normal and infarcted myocardium in the two substrates allowed maximal comparison. Mitochondrial oxygen consumption (microatoms of oxygen consumed per milligram of mitochondrial protein per minute), ADP/O ratio (micromoles of adenosinediphosphate [ADPI phosphorylated per atom of oxygen consumed), and respiratory control index (the ratio of oxygen consumption in the presence of ADP compared to that after ADP) were determined for each tissue and substrate.21 Mitochondrial protein was determined by standard methods, and ADP assays were determined spectrophotometrically. Results Serum samples taken at frequent intervals after the intravenous administration of 0.5 mg. of tritiated digoxin revealed rapid disappearance of digoxin from the serum. Within a 2-hour period after 0.5 mg. of digoxin was administered intravenously, the serum concentration fell from approximately 35 ng. per milliliter at five minutes to 2.1 ng. per milliliter (Fig. 2). It has previously been documented that this rapid fall in serum concentration primarily represents organ distribution and binding,22 and that two hours allow for equilibration of the serum and tissue compartmentally. Myocardial tissue samples taken two hours after the intravenous administration of 0.5 mg. of tritiated digoxin revealed a significant variation in digoxin concentration in and around the infarcted zone (Fig. 3). The concentration of 90 ng. of tritiated digoxin per gram of normal left ventricle and a tissue-to-serum ratio of 43:l is in close agreement with findings previously published by this laboratory.23 The infarcted tissue demonstrated a tissue-to-serum ratio of 12:1, a 3- to 4-fold decrease in tritiated digoxin content as compared to normal myocardium. The
September,
1974, Vol. 88, No. 3
‘l’ritiated
digoxin
I
SE
l.O!
0
I
I
1
I
I5
30
45
60
f
75
6
90
105
120
MINUTES FIQ. 2. Serum disappearance serum concentration (mean horizontal axis.
curve of 3H-digoxin f S.E.M.) is plotted
following the intravenous on the vertical axis, with
injection of 0.5 semilogarithmic
mg. of aH-diguxin. ecale and time
The on the
Table I Wet wei& Experiment No. 1 2 3 4 5 6 I 8 9 10 11 Mean S.E.M. &
Weight OQ..) 13.6 18.0 14.1 23.2 14.5 22.7 12.7 16.4 17.3 25.5 20.9 18.1 1.4
3Hdigoxin dose hg.)
Serum concentration (ng.lml.)
Normal LV
Ischemic LV
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 -
2.32 1.54 1.85 2.22 3.26 1.48 3.38 2.43 2.17 0.90 1.76 2.12 0.67
150.35 84.51 74.28 105.50 114.22 66.10 163.17 70.88 69.75 52.26 29.37 89.13 28.19
130.35 38.35 13.71 39.18 106.26 41.08 99.95 70.34 66.61 33.60 23.52 60.27 19.06
ischemic xone was intermediate between normal and infarct, and a myocardial tissue level of 55 ng. per gram and ratio of 26:l was present. The serum concentration of tritiated digoxin in the cardiac tissues sampled in each of the eleven animal preparation8 is listed in Table I. Tissue samples from the right ventricle, atrium, and pericardial fat were analyzed for
American Heart Journal
Znfareted LV 9.51 18.92 7.71 33.30 48.43 20.54 27.92 26.54 49.82 11.60 1.53 23.98 7.58
fng.lGm.) Pericardial RV
Atrium
fat
92.48 55.32 55.10 67.70 88.16 46.91 127.09 58.26 64.07 45.71 37.42 67.07 21.21
73.98 38.69 44.41 1.06 70.33 28.76 73.23 32.15 38.17 23.85 12.65 45.45 14.37
3.95 2.38 4.66 1.68 4.95 2.01 2.46 0.43 1.50 2.51 kO.51
digoxin content. The normal right ventricle contained less digoxin per gram of tissue than did the normal left ventricle. Atria1 tissue concentrated less digoxin per gram of tissue than did either ventricle. The pericardial fat had a digoxin concentration not significantly different from that of serum. Listed in Table II are the data evaluating the
321
Thompson et al. Table II RCR
@2 Experiment No.
3 5 6 7 10 11 Mean S.E.M. t-
Normal
Infarct
0.4791 0.3979 0.3003 0.2895 0.2890 0.2867 0.3404 0.036
0.0856 0.1649 0.0957 0.0977 0.0823 0.0867 0.1023 0.014
-
NORMAL L.V.
ISCHEMIC L v.
INFARCTED LV
/
Normal
Znfarct
Normal
3.61 3.87 4.02 3.78 4.31 4.24 3.97 0.12
2.12 2.97 2.35 3.03 2.21 2.29 2.50 0.18
2.31 2.38 2.78 2.75 2.89 2.63 2.62 0.10
Infarct
1.87 2.56 2.15 2.09 2.17 2.30 2.19 0.10
specialized equipment. Oxidative phosphorylation determination confirmed the division of tissue into normal and infarcted regions by demonstrating impaired oxygen utilization of mitochondria from infarcted myocardium. Right ventricular myotardium has been shown to concentrate less digoxin than the left ventricle per gram of tissue weight. Atria1 tissue concentrates less digoxin than either ventricle, and pericardial fat appeared to have no tissue-to-serum gradient. The studies revealed a marked disparity of tritiated digoxin distribution in the canine left ventricle after acute myocardial infarction. Homogeneous distribution of tritiated digoxin in the normal canine left ventricle has been demonstrated in previous studies.22,24After intravenous administration, myocardial concentration of tritiated digoxin peaks at one hour and is well maintained for 12 hours in the caninez6; therefore, the two-hour tissue samples should be representative of maximal myocardial concentration. Altered regional blood flow to ischemic myocardium may explain the distribution pattern of tritiated digoxin, as suggested by radioisotope studies utilizing labeled carbonized microspheres26* 27 and i31 I-iodoantipyrine.28 The ischemic myocardium may also show reduced tritiated digoxin uptake due to high extracellular potassium concentrationzs which accumulates from cellular injury and intracellular potassium loss. Normal myocardium has a tissue concentration and tissue-to-serum ratio which closely agrees with previous findings in our laboratory. The myocardial digoxin content decreases progressively in normal, ischemic, and infarcted
I..-Rl”M
PERICARDIAL FAT
Fig. 3. Myocardial concentration of 3H-digoxin. Tissue samples were taken two hours after the intravenous administration of 0.5 mg. of 3H-digoxin. Digoxin concentration in nanograms per gram of wet weight tissue (mean f S.E.M.) is on the ordinate. Concentration in the normal left ventricle is approximately 90 ng. per gram of tissue. The ischemic left ventricle and the infarcted left ventricle contained 54 and 25 ng. per gram of tissue, respectively. The normal right ventricle tritiated digoxin concentration of 64 ng. par gram of tissue is less than the normal left ventricular myocardium. Atria1 tissue contains less tritiated digoxin than either ventricle. Pericardial fat has a concentration of tritiated digoxin equivalent to serum.
metabolic activity of mitochondria from normal and infarcted myocardium in six of the animal preparations. Tissue respiration in malate substrate-as measured by oxygen consumption, respiratory control index, and ADP/O-is signiIicantly reduced in the infarcted tissue, verifying tissue hypoxia and altered mitochondrial integrity in the infarcted zones. Discussion
The normal, ischemic, and infarcted myocardial zones were easily differentiated with a vital dye. This rapid and accurate method required no
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Vitiated
tissue with a marked tritiated digoxin gradient being present between these respective zones. Disparate repolarization and the initiation of reentrant ectopic rhythms have been demonstrated in ischemic myocardium.13v l4 Canine Purkinje fibers exhibit enhanced automaticity in the presence of hypoxia and fiber stretch.30 The similar digitalis effect of enhanced automaticity of Purkinje fibers and facilitation of ectopic rhythms is potentially additive in the ischemic myocardium.31, 32Thus, a marked digoxin gradient between normal, ischemic, and infarcted myocardium may potentiate the appearance of ectopic rhythms from electrically unstable ischemic or infarcted myocardium.
6. 7. 8.
9. 10. 11. 12.
13.
Summary
The role and potential hazards of digitalis glycoside administration in acute myocardial infarction remain controversial. We investigated the concentration of tritiated digoxin in normal, ischemic, and infarcted left ventricular myocardium of the dog after ligation of the anterior interventricular coronary artery. The normal homogeneous distribution of tritiated digoxin in the normal canine left ventricle was altered following acute myocardial infarction. The ischemic and infarcted zones exhibited a marked diminution in digoxin concentration. Oxidative phosphorylation determinations confirmed tissue hypoxia in the infarcted zone. The gradient of digoxin concentration between normal, ischemic, and infarcted zones of myocardium may potentiate the development of an arrhythmia in the electrically unstable infarcted myocardium. We would like to acknowledge the technical assistance of William Lynch, Jacquelyn Gammill, B.S., M.T.(A.S.C.P.), and Joyce Sherwood, B.S., M.T.(A.S.C.P.). Gratitude is expressed to Mrs. Genevieve Lopez for typing of this manuscript. REFERENCES 1.
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