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Effects of intraperitoneal administration of propranolol on the mouse heart
Effects of lntraperitoneat Administration of Propranolol on the Mouse Heart ~isto~he~ical
and Electron microscopic
EDGAR F. ALLIN, MD JANlCE
M. MILLER,
DVM,
PhD
GEORGE G. ROWE, MD JAMES
A.
WILL,
DVM,
PhD
Madison, Wisconsin
Observations
In an earlier inve~~ation, uitrastructural and hlst~hem~al abnormallties were reported In the hearts of mice receiving the beta adrenerglc blocking agent propranolol. Because of the increasing us& of this drug to treat human cardiac disorders, we repeated this investigation. Our findings do not confirm that cardiac lesions are produced by bropranolok No structural or histochemical abnormalities could be related to administration of the drug. We conclude that there is no presently acceptable histologic evidence that propranolol causes myocardial damage.
Because propranolol
is widely used to treat patients
ease,l it is of considerable histochemical and electron drug. In this investigation
importance microscopic
we repeated
Material
From the Car~~vascu~r Research Laboratory, Departments of Anatomy, Medicine and Veterinary Science, University of Wisconsin Medical School, Madison, Wise. 53706. This work was supported in part by Grants H107754 and HL5364 from the National Heart and Lung Institute, Nationalinstitutes of Health, Bethesda, Md. Manuscript accepted October 4, 1973. Address for reprints: George G. Rowe, MD. Cardiovascular Research Laboratory, Rm. 523, 420 North Charter St., Madison, Wise. 53706.
with heart disthat Sun et a1.2 have reported changes in mice receiving this
their study.
and Methods
The study was performed in three parts referred to as experiments I, 2 and 3. The animals were 3 week old Swiss white mice averaging 20 g in weight. Propranolol (Inderale, Ayerst Laboratories) was injected intraperitoneally once daily for 21 days in test animals; all control mice received daily injections of a similar quantity of sterile diluent. On the 22nd day the animals were killed by cervical traction, the thoracic cavity was swiftly exposed and the hearts were promptly treated as described later. Specimens were given code numbers and initial microscopic observations were made without knowledge of the experimental regimen to which they had been subjected. The code was then broken and the sections were reexamined. In experiment I, five mice received 0.05 mg of propranolol daily, five received 0.1 mg of the drug and five served as control animals. All hearts were fixed with glutaraldehyde, portions were removed for electron microscopic studies, then the remaining tissue was embedded in paraffin, sectioned and stained with hematoxylin-eosin. In experiment 2, ten mice were given 0.1 mg of propranolol daily, and five received sham injections for control studies. The hearts of four treated and two control animals were perfused for electron microscopic studies. The remaining nine hearts were frozen for histoehemical processing. In experiment 3, fourteen mice received 0.1 mg of propranolol daily and another 16 mice served as control animals. Both groups gained weight at a similar rate. Three hearts from each group were perfused for electron microscopic studies and three others from each group were frozen for histochemical examination. The remaining 18 hearts were fixed in Bouin’s solution, and 2 mm thick transverse slices of the ventricular region were embedded in paraffin, then sectioned for periodic acid-Schiff and hematoxylin-eosin staining. To insure uniform treatment, one experimental and one control specimen were embedded side by side in each paraffin block and processed together. For electron microscopic studies, 3.5 percent glutaraldehyde in 0.1 molar cacodylate buffer was injected into the left ventricle of the beating heart through a 27 gauge needle. The heart was arrested and removed and the atria plus portions of the ventricles and interventricular septum were immersed in the glutaraldehyde solution for 3 hours, washed overnight in cacodylate buffer, then postfixed for 2 hours in 1 percent osmium tetroxide. After dehydra-
May 1974
The American Journal of CARDIOLOGY
Volume 33
639
Pl?OPf?ANOLOL IN MOUSE HEART-ALLIN
ET AL.
myofibrillar tase.s
(calcium-activated)
adenosine
triphospha-
Sesults Electron
microscopy:
No consistent
differences
were observed between sections from tissues of propranolol-treated and control mice. Although ultrastructural anomalies were sometimes noted, they occurred with equal frequency in the control and treated groups. Occasionally, isolated mitochondria were swollen and cristae were disrupted or absent (Fig. 1, top). Less commonly there was a separation of myofibrils at the region of the intercalated disc. There was considerable variation in the amount of glycogen granules among sections but no difference between propranolol-treated and control groups. The great majority of sections of treated hearts appeared entirely normal (Fig. 1, bottom). Histochemistry: Experimental animals could not be differentiated from control animals after repeated careful scrutiny. Total glycogen content varied from heart to heart but not in any systematic way. There were occasional features that might be taken as abnormal but they were no more frequent or marked in experimental than in control animals (Fig. 2). Most were probably artifacts, which are unavoidable in histochemical studies. Reduced oxidative enzyme activity was often evident in the region of intercalated discs in all hearts. No differences in blood vessel caliber or staining were evident. No pronounced 5’-nucleotidase activity was observed in myofibers of any of the hearts. Overall enzyme activity levels were similar in control and treated hearts.
FIGURE 1. Electron micrographs of left ventricular tissue of mice from experiment 3. Top, control heart. Many mitochondria are swollen and have disrupted cristae. Bottom, treated heart (0.1 mg of propranolol). No ultrastructural abnormalities are present. Top, X 13,500; bottom, X 10,000, both reduced by 28 percent.
tion, tissue blocks were embedded in an Epon-Araldite plastic mixture.” Thin sections were stained in 25 percent uranyl acetate4 and lead citrate5 and examined in a Hitachi HU II-B electron microscope. Multiple samples, both adluminal and deeper, were examined from every heart, with several from each atrium, each ventricle and the interventricular septum. For histochemical processing, hearts of simultaneously killed mice were swiftly excised without pinching or other mechanical injury, assembled in uniform orientation on a copper planchette and frozen in liquid nitrogen or isopentane slurry. Each planchette held one control and one or two treated hearts. Ten h coronal sections were cut in a cryostat microtome, with at least six whole heart sections including all chambers for each of the following techniques: (1) Sudan black B for lipids’s; (2) periodic acid-Schiff for glycogen, with diastase controls6 (fixation by formaldehyde fumes and Carnoy’s solution); and (3) enzyme activity determinations: succinic dehydrogenase,6 nicotinamide-adenine dinucleotide, reduced form (NADH) diaphorase,s beta-hydroxybutyric dehydrogenase,” 5’-nucleotidase7 and
640
May 1974
The American Journal of CARDtOLDGY
Volume 33
Discussion In our study, in contrast to that of Sun et a1.,2 it was not possible to distinguish the hearts of control and propranolol-treated mice on the basis of ultrastructural or histochemical characteristics. Although we observed occasional features resembling those interpreted as lesions in the earlier study, they were neither more abundant nor more conspicuous in treated than in control animals. We interpret these features as artifactual or, in some instances, normal. If this interpretation also holds for the “lesions” of Sun et al., as seems probable, then an explanation must be sought for the reported association with administration of propranolol. Systematic artifacts could result if the control and experimental tissues were handled differently although the reported correlation between the dose of propranolol and the degree of abnormality is difficult to explain on this basis. Alternatively, the drug may have produced true lesions that were overlooked or failed to appear in our study. Since only small samples of tissue can be inspected at sufficient magnification for ultrastructural resolution in the electron microscopic studies, sporadic abnormalities might escape detection, but it is unlikely that frequent or extensive lesions were missed. As for the possibility that true lesions appeared in the earlier study but not in our investigation, various agencies could be responsible. Mice of different age or strain might be differently affected
FSWRANDLGL IN MfBBff IWtRT-AWN
IRE 2. Hi&chemical pseudolesions. All sections are from the left ventrkzle, and all except one (A) are stained for succfnlc dehydrogenase activity. A, control heart: NADH diaphorase activity. Cross section showing areas of bw intensity (arrows) resembling the “loss of mite&or&a” ln Fiie 1 of Sun et al.* B, control heart: ~~~ sectbn with discontinuityof fibers (arrows) similar to the “fragmentation”in Figure 3 of Sun et al.* This is actually a planeof-section effect. C. control heart: longitudinal section showing areas of hi intenshy (arrowe) resemblfng the “clumping of the rn~” in Figure 3 of Sun et al.* D, treated heart (0.1 mg propranolol): representative longltudlnal section lacking evident abnormality. A to C X 600, D X
ET AL.
c
by the drug, but in both studies the animals were of the same strain and of similar initial weight. Some aspect of the environment, health or handling of the mice might conceivably have resulted in enhanced susceptibility to the drug in the earlier study. Sun et al. did not state whether their control mice were sham-injected as ours were. If not, differential stress might have been a factor. A difference in the potency or purity of the drug is unlikely since the source was the same. Similarly, the route of administration and largest dose used were alike in both studies. Sun et al. did not indicate how long after the last injection their mice were killed. This might be a significant variable. In the earlier study the histochemical results were apparently dissimilar for different mitochondrial enzymes. Marked lesions were found for succinic dehydrogenase and “lactic dehydrogenase” (the technique primarily reveals NADH diaphorase activity), yet the authors reported “relatively little change in cytochrome oxidase and alpha-glycerophosphate dehydrogenase activity” and only a “generally increased” activity of beta-hydroxybutyric dehydrogenase. One would expect all mitochondrial enzymes to be affected in areas of mitochondrial abnormality. Virtually identical results were obtained in our study for the three oxidative enzymes examined (succinic dehydrogenase, NADH diaphorase and beta-hydroxybutyric dehydrogenase).
To minimize the risk of interpretive bias, our initial observations were made without foreknowledge of the treatment history of the specimens. To minimize fixation artifacts in the electron microscopic aspect of the study, we perfused the hearts with glutaraldehyde. Sun et al. excised multiple samples and fixed these by immersion in osmium tetroxide. The delay involved in such a procedure is undesirable in a metabolically active, aerobic tissue. Also, Sun et al. froze for histochemical processing heart tissue remaining after excision of samples for electron microscopic studies, with both delay and mechanical trauma. Although artifacts can never entirely be eliminated, we consider our procedures less likely to produce them. Clinical implications: Although a negative proposition cannot be proved, our results do not support the contention that propranolol in the doses used produces electron microscopic, histochemical or other histologic evidence of cardiotoxicity in mice. This is reassuring in view of the widespread utilization of propanolol in treatment of cardiac arrhythmias,g angina pectorislO and hypertension.ll As recommended by Sun et a1.,2 careful observation must be continued in human subjects receiving propranol01. Clinical surveillance is justified because of the known side effects of this drug, but our study provides no evidence of myocardial damage.
References 1. Pitt 6,
Ross RS: Beta adrenergic blockade in cardiivascular therapy. Mod Concepts Cardiivasc Dis 3647-54, 1969 2. Sun SC, Burch GE, GePaequale NP: Hlstochemical and electron microscope study of heart muscle after beta-adrenergic blockade. Am Heart J 74~340-350, 1967
3. ~a~ HH: f%stic embedding mixtures for use in electron mfcroscope. Stein Technol39: 11 1- 114, 1964 4. Stempak JF, Ward RT: An improved staining method for electron microscopy. J Cell Bbl22:697-70 1, 1964 5. Aeynotds ES: The use of lead &rate at high pH as an electron-
May 1974
The Am&can
Journal ot CARDtGLGGY
Votume 33
fM1
F’ROPRANDLDL IN w
r
HiART-ALLiN
ET AL.
stain in efectron mkroscopy.
J Cefl WI
17208-212,
6. LMe RDr Histopathobgk Technique and Practicpll Htstochemistry, third editlon, New York, McGraw-Hill, 1965, p 196-199, 38 l-384,468,496-49? 7. Suka T, Andweon P& Histochemistry. New York, Harper & Row, 1903, p 248 8. Quth L, Sam&a FJ: Qualitative differences between actomyosln ATPase of slow and fast mammatian muscte. Exp Neurol
642
May 1974
The Amerksn
Journal of CARDIDLDGY
Volume 33
25: 138, 1969 9. Reyeetds SW Jr: Beta blocking agents in the management of card& arrhythmias. Certatrlcs 26:150-161, 1971 10. Aronow WS: The medical treatment of angina pectoris. VI. Propranotot as an annual drug. Am Heart J 84:?06-709,1972 11. Richardson DW, Fround J, Gear AS, et al: Effect of propranolol on elevated arterial blood pressure. Circulation 37534-542, 1966
and Electron microscopic
EDGAR F. ALLIN, MD JANlCE
M. MILLER,
DVM,
PhD
GEORGE G. ROWE, MD JAMES
A.
WILL,
DVM,
PhD
Madison, Wisconsin
Observations
In an earlier inve~~ation, uitrastructural and hlst~hem~al abnormallties were reported In the hearts of mice receiving the beta adrenerglc blocking agent propranolol. Because of the increasing us& of this drug to treat human cardiac disorders, we repeated this investigation. Our findings do not confirm that cardiac lesions are produced by bropranolok No structural or histochemical abnormalities could be related to administration of the drug. We conclude that there is no presently acceptable histologic evidence that propranolol causes myocardial damage.
Because propranolol
is widely used to treat patients
ease,l it is of considerable histochemical and electron drug. In this investigation
importance microscopic
we repeated
Material
From the Car~~vascu~r Research Laboratory, Departments of Anatomy, Medicine and Veterinary Science, University of Wisconsin Medical School, Madison, Wise. 53706. This work was supported in part by Grants H107754 and HL5364 from the National Heart and Lung Institute, Nationalinstitutes of Health, Bethesda, Md. Manuscript accepted October 4, 1973. Address for reprints: George G. Rowe, MD. Cardiovascular Research Laboratory, Rm. 523, 420 North Charter St., Madison, Wise. 53706.
with heart disthat Sun et a1.2 have reported changes in mice receiving this
their study.
and Methods
The study was performed in three parts referred to as experiments I, 2 and 3. The animals were 3 week old Swiss white mice averaging 20 g in weight. Propranolol (Inderale, Ayerst Laboratories) was injected intraperitoneally once daily for 21 days in test animals; all control mice received daily injections of a similar quantity of sterile diluent. On the 22nd day the animals were killed by cervical traction, the thoracic cavity was swiftly exposed and the hearts were promptly treated as described later. Specimens were given code numbers and initial microscopic observations were made without knowledge of the experimental regimen to which they had been subjected. The code was then broken and the sections were reexamined. In experiment I, five mice received 0.05 mg of propranolol daily, five received 0.1 mg of the drug and five served as control animals. All hearts were fixed with glutaraldehyde, portions were removed for electron microscopic studies, then the remaining tissue was embedded in paraffin, sectioned and stained with hematoxylin-eosin. In experiment 2, ten mice were given 0.1 mg of propranolol daily, and five received sham injections for control studies. The hearts of four treated and two control animals were perfused for electron microscopic studies. The remaining nine hearts were frozen for histoehemical processing. In experiment 3, fourteen mice received 0.1 mg of propranolol daily and another 16 mice served as control animals. Both groups gained weight at a similar rate. Three hearts from each group were perfused for electron microscopic studies and three others from each group were frozen for histochemical examination. The remaining 18 hearts were fixed in Bouin’s solution, and 2 mm thick transverse slices of the ventricular region were embedded in paraffin, then sectioned for periodic acid-Schiff and hematoxylin-eosin staining. To insure uniform treatment, one experimental and one control specimen were embedded side by side in each paraffin block and processed together. For electron microscopic studies, 3.5 percent glutaraldehyde in 0.1 molar cacodylate buffer was injected into the left ventricle of the beating heart through a 27 gauge needle. The heart was arrested and removed and the atria plus portions of the ventricles and interventricular septum were immersed in the glutaraldehyde solution for 3 hours, washed overnight in cacodylate buffer, then postfixed for 2 hours in 1 percent osmium tetroxide. After dehydra-
May 1974
The American Journal of CARDIOLOGY
Volume 33
639
Pl?OPf?ANOLOL IN MOUSE HEART-ALLIN
ET AL.
myofibrillar tase.s
(calcium-activated)
adenosine
triphospha-
Sesults Electron
microscopy:
No consistent
differences
were observed between sections from tissues of propranolol-treated and control mice. Although ultrastructural anomalies were sometimes noted, they occurred with equal frequency in the control and treated groups. Occasionally, isolated mitochondria were swollen and cristae were disrupted or absent (Fig. 1, top). Less commonly there was a separation of myofibrils at the region of the intercalated disc. There was considerable variation in the amount of glycogen granules among sections but no difference between propranolol-treated and control groups. The great majority of sections of treated hearts appeared entirely normal (Fig. 1, bottom). Histochemistry: Experimental animals could not be differentiated from control animals after repeated careful scrutiny. Total glycogen content varied from heart to heart but not in any systematic way. There were occasional features that might be taken as abnormal but they were no more frequent or marked in experimental than in control animals (Fig. 2). Most were probably artifacts, which are unavoidable in histochemical studies. Reduced oxidative enzyme activity was often evident in the region of intercalated discs in all hearts. No differences in blood vessel caliber or staining were evident. No pronounced 5’-nucleotidase activity was observed in myofibers of any of the hearts. Overall enzyme activity levels were similar in control and treated hearts.
FIGURE 1. Electron micrographs of left ventricular tissue of mice from experiment 3. Top, control heart. Many mitochondria are swollen and have disrupted cristae. Bottom, treated heart (0.1 mg of propranolol). No ultrastructural abnormalities are present. Top, X 13,500; bottom, X 10,000, both reduced by 28 percent.
tion, tissue blocks were embedded in an Epon-Araldite plastic mixture.” Thin sections were stained in 25 percent uranyl acetate4 and lead citrate5 and examined in a Hitachi HU II-B electron microscope. Multiple samples, both adluminal and deeper, were examined from every heart, with several from each atrium, each ventricle and the interventricular septum. For histochemical processing, hearts of simultaneously killed mice were swiftly excised without pinching or other mechanical injury, assembled in uniform orientation on a copper planchette and frozen in liquid nitrogen or isopentane slurry. Each planchette held one control and one or two treated hearts. Ten h coronal sections were cut in a cryostat microtome, with at least six whole heart sections including all chambers for each of the following techniques: (1) Sudan black B for lipids’s; (2) periodic acid-Schiff for glycogen, with diastase controls6 (fixation by formaldehyde fumes and Carnoy’s solution); and (3) enzyme activity determinations: succinic dehydrogenase,6 nicotinamide-adenine dinucleotide, reduced form (NADH) diaphorase,s beta-hydroxybutyric dehydrogenase,” 5’-nucleotidase7 and
640
May 1974
The American Journal of CARDtOLDGY
Volume 33
Discussion In our study, in contrast to that of Sun et a1.,2 it was not possible to distinguish the hearts of control and propranolol-treated mice on the basis of ultrastructural or histochemical characteristics. Although we observed occasional features resembling those interpreted as lesions in the earlier study, they were neither more abundant nor more conspicuous in treated than in control animals. We interpret these features as artifactual or, in some instances, normal. If this interpretation also holds for the “lesions” of Sun et al., as seems probable, then an explanation must be sought for the reported association with administration of propranolol. Systematic artifacts could result if the control and experimental tissues were handled differently although the reported correlation between the dose of propranolol and the degree of abnormality is difficult to explain on this basis. Alternatively, the drug may have produced true lesions that were overlooked or failed to appear in our study. Since only small samples of tissue can be inspected at sufficient magnification for ultrastructural resolution in the electron microscopic studies, sporadic abnormalities might escape detection, but it is unlikely that frequent or extensive lesions were missed. As for the possibility that true lesions appeared in the earlier study but not in our investigation, various agencies could be responsible. Mice of different age or strain might be differently affected
FSWRANDLGL IN MfBBff IWtRT-AWN
IRE 2. Hi&chemical pseudolesions. All sections are from the left ventrkzle, and all except one (A) are stained for succfnlc dehydrogenase activity. A, control heart: NADH diaphorase activity. Cross section showing areas of bw intensity (arrows) resembling the “loss of mite&or&a” ln Fiie 1 of Sun et al.* B, control heart: ~~~ sectbn with discontinuityof fibers (arrows) similar to the “fragmentation”in Figure 3 of Sun et al.* This is actually a planeof-section effect. C. control heart: longitudinal section showing areas of hi intenshy (arrowe) resemblfng the “clumping of the rn~” in Figure 3 of Sun et al.* D, treated heart (0.1 mg propranolol): representative longltudlnal section lacking evident abnormality. A to C X 600, D X
ET AL.
c
by the drug, but in both studies the animals were of the same strain and of similar initial weight. Some aspect of the environment, health or handling of the mice might conceivably have resulted in enhanced susceptibility to the drug in the earlier study. Sun et al. did not state whether their control mice were sham-injected as ours were. If not, differential stress might have been a factor. A difference in the potency or purity of the drug is unlikely since the source was the same. Similarly, the route of administration and largest dose used were alike in both studies. Sun et al. did not indicate how long after the last injection their mice were killed. This might be a significant variable. In the earlier study the histochemical results were apparently dissimilar for different mitochondrial enzymes. Marked lesions were found for succinic dehydrogenase and “lactic dehydrogenase” (the technique primarily reveals NADH diaphorase activity), yet the authors reported “relatively little change in cytochrome oxidase and alpha-glycerophosphate dehydrogenase activity” and only a “generally increased” activity of beta-hydroxybutyric dehydrogenase. One would expect all mitochondrial enzymes to be affected in areas of mitochondrial abnormality. Virtually identical results were obtained in our study for the three oxidative enzymes examined (succinic dehydrogenase, NADH diaphorase and beta-hydroxybutyric dehydrogenase).
To minimize the risk of interpretive bias, our initial observations were made without foreknowledge of the treatment history of the specimens. To minimize fixation artifacts in the electron microscopic aspect of the study, we perfused the hearts with glutaraldehyde. Sun et al. excised multiple samples and fixed these by immersion in osmium tetroxide. The delay involved in such a procedure is undesirable in a metabolically active, aerobic tissue. Also, Sun et al. froze for histochemical processing heart tissue remaining after excision of samples for electron microscopic studies, with both delay and mechanical trauma. Although artifacts can never entirely be eliminated, we consider our procedures less likely to produce them. Clinical implications: Although a negative proposition cannot be proved, our results do not support the contention that propranolol in the doses used produces electron microscopic, histochemical or other histologic evidence of cardiotoxicity in mice. This is reassuring in view of the widespread utilization of propanolol in treatment of cardiac arrhythmias,g angina pectorislO and hypertension.ll As recommended by Sun et a1.,2 careful observation must be continued in human subjects receiving propranol01. Clinical surveillance is justified because of the known side effects of this drug, but our study provides no evidence of myocardial damage.
References 1. Pitt 6,
Ross RS: Beta adrenergic blockade in cardiivascular therapy. Mod Concepts Cardiivasc Dis 3647-54, 1969 2. Sun SC, Burch GE, GePaequale NP: Hlstochemical and electron microscope study of heart muscle after beta-adrenergic blockade. Am Heart J 74~340-350, 1967
3. ~a~ HH: f%stic embedding mixtures for use in electron mfcroscope. Stein Technol39: 11 1- 114, 1964 4. Stempak JF, Ward RT: An improved staining method for electron microscopy. J Cell Bbl22:697-70 1, 1964 5. Aeynotds ES: The use of lead &rate at high pH as an electron-
May 1974
The Am&can
Journal ot CARDtGLGGY
Votume 33
fM1
F’ROPRANDLDL IN w
r
HiART-ALLiN
ET AL.
stain in efectron mkroscopy.
J Cefl WI
17208-212,
6. LMe RDr Histopathobgk Technique and Practicpll Htstochemistry, third editlon, New York, McGraw-Hill, 1965, p 196-199, 38 l-384,468,496-49? 7. Suka T, Andweon P& Histochemistry. New York, Harper & Row, 1903, p 248 8. Quth L, Sam&a FJ: Qualitative differences between actomyosln ATPase of slow and fast mammatian muscte. Exp Neurol
642
May 1974
The Amerksn
Journal of CARDIDLDGY
Volume 33
25: 138, 1969 9. Reyeetds SW Jr: Beta blocking agents in the management of card& arrhythmias. Certatrlcs 26:150-161, 1971 10. Aronow WS: The medical treatment of angina pectoris. VI. Propranotot as an annual drug. Am Heart J 84:?06-709,1972 11. Richardson DW, Fround J, Gear AS, et al: Effect of propranolol on elevated arterial blood pressure. Circulation 37534-542, 1966