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Determinants of resistance to the cardiotoxicity of isoproterenol in rats
TOXICOLOGY
AND APPLIED PHARMACOLOGY
Determinants
of Resistance
XAVIERJOSEPH,*
69, 199-205 (1983)
to the Cardiotoxicity
of lsoproterenol
SHERMAN BLOOM,? GORDONPLEDGER,*
*National Center for Drugs and Biologics, Food iDepartment of Pathology, George Washington
Received
November
in Rats
ANDTIBORBALAZS*
and Drug Administration, Washington, D.C. 20204 and University Medical Center, Washington, D.C. 20037
4. 1982: accepted
February
9, 1983
Determinants of Resistance to the Cardiotoxicity of Isoproterenol in Rats. JOSEPH, X., BLOOM, S., PLEDGER, G., AND BALAZS, T. (1983). Toxicol. Appl. Pharmacol. 69, 199-205. Induction of myocardial necrosis by isoproterenol produces resistance to the necrogenic effects of subsequent doses of the drug. A series of experiments were performed to further define the determinants of resistance. Myocardial necrosis was induced in male Sprague-Dawley rats by SCinjection of isoproterenol at 50 pglkg daily for 10 consecutive days or as a single dose at 50, 5, or 0.5 pg/ kg. These preconditioning doses were followed, at various times, by a challenge dose of 50 ccgl kg. The rats were killed 48 hr after the challenge dose, and their hearts were analyzed morphometrically to determine the amount of acute necrosis and scarring. The amount of scar tissue was a reflection of necrosis caused by the preconditioning dose whereas acute necrosis reflected response to the challenge dose. Resistance occurred and lasted longer than 19 to 20 weeks after both single or multiple isoproterenol injections of 50 ccg/kg,but it was not observed 5 days after administration of a single preconditioning dose.Isoproterenol at 0.5 pg/kg produced only very minimal or no myocardial necrosis and did not produce resistance. The resistance was not dependent on the size of the area of necrosis produced during the preconditioning period, showing that it was not due to destruction of all vulnerable muscle by the preconditioning dose(s). The preexistence of lesions, however, was necessary for the development of resistance. It is concluded that development of resistance to the necrogenic effects of isoproterenol reflects an adaptive alteration in the myocardium which survives after a necrogenic dose.
There are reports about the lack of sensitivity of rat myocardium to the necrosis-inducing effects of isoproterenol after an initial insult by either drugs (Balazs et al., 1972) or other means such as coronary artery ligation (Selye et al., 1960; Dusek et al., 1970) or strenuous muscular exercise (Bajusz, 1965). This phenomenon has usually been referred to as myocardial resistance or protection (Dusek et al., 197 la). Selye et al. (1960) showed that ligation of the left coronary artery in rats provided resistance against isoproterenol-induced myocardial necrosis when the drug was given a few days after ligation. Dusek et al. (1970) found that the protective effect was independent of the location of initial necrosis in the heart; they also reported that the myo-
cardial resistance was maximal 48 hr after coronary ligation and then gradually declined so that the myocardium was again sensitive to isoproterenol-induced necrosis 3 weeks after the ligation (Dusek et al., 1971a). Balazs et al. ( 1972) found that myocardial resistance to isoproterenol challenge occurred in rats pretreated with isoproterenol for 2 consecutive days and that the sensitivity to isoproterenol had returned 11 weeks after the initial treatment. Our present investigation was undertaken to study the development of resistance to isoproterenol challenge and its duration in rats which were pretreated with daily injections of isoproterenol for 10 consecutive days or with a single injection. In addition, experiments 199
0041-008X/83 Copyright All n&s
$3.00
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
200
JOSEPH ET AL.
were performed to determine whether the development of resistance is related to elimination of the most sensitive subendocardial area by the preceding insults and whether the initial development of myocardial lesions is a prerequisite for the resistance phenomenon.
Histopathology
In all three experiments, animals were killed by CO2 inhalation and were necropsied. Hearts were examined in situ, removed, rinsed in isotonic saline, and again examined for gross lesions. Serial transverse sections, 2-mm thick, were made from the apex to the base of the heart; these sections included both right and left ventricles. For microscopic studies, the second and fourth sections, counting from the apex, were fixed in 4% buffered formMETHODS aldehyde and processed by routine histologic procedures. Sections were cut at a normal thickness of 5 Mm and Male Sprague-Dawley rats weighing 400 to 500 g (3 stained by Masson’s trichrome method; they were asto 4 months old) were obtained from Charles River Breedsigned random numbers to ensure unbiased analysis. ing Laboratories, Wilmington, Mass. and were housed Morphometric analysis was performed by projecting the image of the slide onto graph paper and counting the two in a cage with free accessto feed and water. frequency with which normal or abnormal areas of the myocardium overlapped points of intersection on the paExperiment I per. The apical-most section of ventricular myocardium was used for grading. Three morphological features were used for evaluation purposes: (1) normal tissue, which One hundred forty rats were randomly assigned to two stained red, (2) acutely necrotic tissue with inflammatory equal groups. Rats of the first group were given DL-iSOproterenol HCl (Sigma Chemical Co., St. Louis, MO.) at cells, edema fluid, and necrotic muscle fibers, which stained 50 &kg sc for 10 consecutive days (Days 1 to 10) and light blue; and (3) scars with dense collagen, which stained rats of the second group received saline. On each of Days dark blue. The percentage of the area represented by each 9 and 10, 10 rats from the second group were treated SC of these features was calculated. with isoproterenol at 50 pg./kg, and these rats and 10 rats from the first group were killed on Day 11. Ten rats that had received only saline were also killed on the same day. Statistical Analysis Of the remaining rats, 10 from each group were challenged Results were analyzed statistically by Wilcoxon onewith isoproterenol at 50 &kg sc for 2 days beginning on Days 2 1, 37, 7 1, or 14 1 and were killed 24 hr after the sample test procedures. Values are expressed as sample median lesion scores (SMLS) with 95% confidence intersecond challenge dose. vals for the median, unless otherwise indicated. (These values are actually pseudomedians as defined by Hoyland (1965), but are very close to median values for data of Experiment II this nature.) Each group was compared with its concurrent control group for significance by Wilcoxon rank-sum Seventy rats were given a single sc injection of isoproterenol HCI at 50 &kg (Day 1) and another group of 70 tests. The differences were considered significant at p < 0.05. rats received saline. Eight rats from each group were killed 48 hr later. On Days 5, 12, 21, 43, 82, and 134, 8 to 10 rats each from both the isoproterenol- and saline-treated RESULTS groups were challenged with a single SCinjection of isoproterenol (50 &kg) and were killed 48 hr later.
Experiment Experiment Ill Seventy-two rats were assigned to three equal groups. Rats of the first and second groups were given a single sc injection of isoproterenol HCI at 5.0 and 0.5 &kg, respectively, and rats of the third group were given saline. Eight animals from each of the three groups were killed 48 hr later. Of the remaining rats, eight from each group were challenged with a single injection of isoproterenol (50 &kg sc) on Days 5 or 21 and were killed 48 hr thereafter.
I
Twelve of the seventy rats treated with isoproterenol for 10 consecutive days died during the treatment period. A total of ten rats from this pretreated group also died at various intervals during isoproterenol challenge. From the control group pretreated with saline and then challenged with isoproterenol, a total of 20 rats died during different intervals. Gross examination of the hearts of rats that
MYOCARDIAL
RESISTANCE
were treated with isoproterenol for 10 days revealed marked pale greyish areas of lesions at the apical region of the ventricular walls. Gross heart lesions were also observed in most of the animals that had received only 2 days of treatment with isoproterenol. Histologically, the animals treated for 2 days showed fresh myocardial necrosis, and those treated for 10 days showed lesions in various stages of healing, including well-healed scars. The isoproterenol-induced lesions have been previously described in detail (Bloom, 1981). The lesions were mainly seen at the apex, left ventricular walls, and papillary muscles; most were localized in the subendocardial layer of the free wall and septum of the left ventricle. Fresh lesions consisted of small foci or multiple areas of muscle necrosis associated with interstitial edema and inflammatory cell infiltration. The inflammatory infiltrate consisted mainly of mononuclear cells and rarely of neutrophils. The necrotic muscle cells
TO ISOPROTERENOL
201
showed a loss of cross striations and the presence of prominent hypercontraction bands. The right ventricular wall was not usually involved. Healing lesions consisted of varying amounts of collagen and fibroblasts with or without chronic inflammatory cells, depending on the age of the lesion. Old lesions consisted of dense scars, with no inflammatory cells and a few fibroblasts. Some of these changes are shown in Fig. 1. In rats that received both the initial or preconditioning dose and a subsequent challenge dose, the scar represented necrosis caused by the preconditioning dose and the acute necrosis, if any, reflected response to the challenge dose. Table 1 summarizes the extent of myocardial lesions in rats pretreated with isoproterenol or saline consecutively for 10 days and challenged at different intervals. The amount of fresh myocardial necrosis after 10 days of treatment with isoproterenol was minimal (SMLS 0.3 compared with 11.4 for acute con-
FIG. 1. Photomicrograph of left ventricular myocardium of rat given 50 pg isoproterenol HCl/kg once daily for 10 days, challenged with the same dose on days 141 and 142, and killed on day 143. Dense scar (a) is present, as well as acute necrosis (b) and normal muscle (c). Masson’s trichrome stain, 300X.
202
JOSEPH ET AL. TABLE 1 EXTENT
OF MY~CARDIAL FOR
LESIONS
10 CONSECUTIVE
IN RATS
DAYS
AND
PRETREATED CHALLENGED
WITH
ISOPROTERENOL
WITH
IS0
AT VARIOUS
HCI
(ISO)
OR SALINE
INTERVALS
Sample median lesion scores (%) with 95% confidence interval” Days of challenge (ISO 50 a/kid
Day killed
IS0 pretreatment”
Saline pretreatmenth
Acute necrosis
Scar
Acute necrosis
No challenge 9, 10 21,22 37, 38 71, 72 141, 142
11 11 23 39 73 143
0.3 (O-6.2) 0 (O-0.2) 0.8 (O-4.2) 1.6 (O-3.4) 3.7 (2.0-4.6)
16. I (9.4-22.5) 18.6 (8.2-34.8) 10.9 (6.3-17.3) 16.1 (10.9-22.0) 15.6 (5.9-25.1)
0.3 (O-0.7) 11.4 (6.6-16.9) 14.0 (6.0-22.3)’ 11.2 (5.6-19.1)’ 8.4 (2.1-23.1)’ 20.2 (6.8-34.2)‘:d
Scar 0.3 0 0 0.2 0 0.5
(O- 1.1) (O-0.2) (O-0.1)’ (O-0.4)’ (O-1.7)’ (0.3-I.o)c”
’ Each value represents the sample median lesion score from 5 to 10 hearts. ’ Pretreatment with IS0 at 50 wg/kg SCor saline was given for 10 consecutive days (Days 1 to 10). ’ Significantly different from value of concurrent ISO-pretreated group (p i 0.05). d 88% confidence interval.
trols), and the amount of old lesions was extensive (SMLS 16.1 compared with zero for acute controls). The saline-treated rats usually did not have any lesions. The amount of acute necrosis in pretreated animals was significantly less than that in their concurrent controls at each of the four challenge periods, i.e., on Days 21, 37, 71, and 141. The amount of scarring was significantly greater in isoproterenol-pretreated groups at each stage, compared with their respective controls. Even though there appeared to be a trend for increased fresh myocardial necrosis in the isoproterenol-pretreated group with passage of time, i.e., from Days 2 1 to 14 1, the values for the respective control groups were always significantly higher. Experiment
II
In Experiment II, 1 of 70 rats died on the first day of isoproterenol treatment, and a total of 5 from the pretreated group died during isoproterenol challenge at various intervals. From the saline-treated group, only two rats died during isoproterenol challenge. The extent of the myocardial lesions in various groups of rats is summarized in Table 2. The
lesions were less severe than those occurring in the first experiment. Rats that were pretreated with a single SCinjection of isoproterenol and challenged on Days 12, 21, 82, or 134 had significantly lower scores of acute myocardial lesions (p < 0.05) compared with their concurrent controls pretreated with saline and challenged with isoproterenol. The amount of scarring was significantly greater in the above groups than in their respective controls. The fresh and old lesion scores in rats pretreated with isoproterenol and challenged on Days 5 and 43 were not significantly different from the control values. Experiment
III
All the rats in Experiment III survived. Table 3 summarizes the extent of myocardial lesions in rats pretreated with a single SCinjection of low doses of isoproterenol (5.0 or 0.5 pg/kg) or saline and then challenged with doses 10 to 100 times higher. Single SCinjections of isoproterenol at 5.0 pg/kg produced significantly more acute lesions (SMLS 1.2) compared with the group that received 0.5 cLg/ kg (SMLS 0.1). Saline-treated rats had no lesions. The acute lesion scores of rats that were
MYOCARDIAL
RESISTANCE
203
TO ISOPROTERENOL
TABLE 2 EXTENT
OF MY~CARDIAL
LESIONS IN RATS PRETREATED WITH A SINGLE DOSE OF LWPROTERENOL OR SALINE AND CHALLENGED WITH IS0 AT VARIOUS INTERVALS
HCl (ISO)
Sample median lesion scores (a) with 95% confidence interval’ Day of challenge (ISO 50 /.&kg)
Day killed
IS0 pretreatment * Acute necrosis
No challenge 5 12 21 43 82 134
3 7 14 23 45 84 136
6.0 (3.2-10.0) 1.3 (0.8-2.8) 0.7 (0.2-1.9) 0.6 (0.1-1.5) 2.4 (1.5-3.9) 0.9 (0.2-l .8) 0.2 (0-2.4)d
Saline pretreatment*
Scar 0.5 4.0 3.8 5.0 2.7 3.0 4.1
Acute necrosis
(0.1-0.8) (0.2-13.1) (2.4-7.6) (2.0-8.2) (0.8-5.6) (1.7-8.9) (2.7-6.4)d
0.4 3.2 4.0 5.4 4.2 6.3 5.4
(O-2.7) (0.5-5.8) (2.7-10.8)’ (1.8-8.8)C (2.0-12.4) (1.1-13.2)’ (1.4-8.1)’
scar 0.5 (0.2-0.8) 1.0 (0.5-1.6) 0.3 (0. I-O.S)C 0.8 (0.3-1.6)’ 0.8 (0.4-l .2) 1.5 (0.7-2.4)’ 1.8 (o-4.0)’
’ Each value represents the sample median lesion score from 5 to 10 hearts. ’ Pretreatment with a single sc dose of IS0 at 50 &kg or saline was given on Day 1. ’ Significantly different from value for concurrent ISO-pretreated group (p < 0.05). d 98% confidence interval.
pretreated with a single SC injection of isoproterenol at 5.0 or 0.5 &kg and challenged with a single dose at 50 pg/kg on Day 5 were not significantly different from the control values. The acute lesion score in rats pretreated with isoproterenol at 5.0 &kg and challenged with 50 bg/kg on Day 21 was significantly less (SMLS 1.5) compared with rats pretreated with 0.5 pg/kg and challenged with 50 pg/kg (SMLS 7.5). Even though the acute
lesion score was much lower in the group pretreated with 5.0 pg/kg (SMLS 1.5) than in the saline-pretreated group (SMLS 4.7), the values were not statistically different. DISCUSSION The results of the present study indicate that pretreatment with isoproterenol decreases the sensitivity of rat myocardium to
TABLE 3 EXTENT OF MY~CARDIAL LESIONS IN RATS PRETREATED WITH Low DOSES OF ISOPROTERENOL OR SALINE AND CHALLENGED WITH IS0 AT VARIOUS INTERVALS
HCl (ISO)
Sample median lesion scores (%) with 95% confidence interval” IS0 pretreatment
b
5.0 a/k Day of challenge (ISO 50 fig/kg)
Day killed
No challenge 5 21
3 7 23
Acute necrosis 1.2 (0.149) 2.3 (1.1-8.7) 1.5 (0.3-4.0)
Saline pretreatmentb
0.5 e/kg Scar
0.7 (0.4-1.2) 1.6 (0.8-2.5) 1.0 (0.7-1.4)
Acute necrosis 0.1 (O-1.0)’ 3.9 (1.6-10.9) 7.5 (1.8-13.4)’
SCX 0.6 (0.3-1.0) 0.6 (0.2-1.4) 0.7 (0.4-0.9)
a Each value represents the sample median lesion score from 6 to 10 rats. b Pretreatment with a single sc dose of IS0 at 5.0 or 0.5 &kg or saline was given on Day 1. ’ Significantly different from value for rats pretreated with 5.0 &kg ISO.
Acute necrosis 0 5.7 (1.4-10.5) 4.7 (1.0-14.9)
scar 0.9 (0.4-1.8) 0.7 (O-2.2) 1.1 (0.7-2.9)
204
JOSEPH ET AL.
the lesion-inducing effects of a subsequent challenge; i.e., after an initial injury, the myocardium develops a resistance to further lesion-inducing insults. This finding is consistent with the findings of other investigators (Balazs et al., 1972; Maramaa and Liiv, 1973). The data also show that the sensitivity to isoproterenol of rats pretreated with either single or multiple doses of isoproterenol does not return even 19 to 20 weeks after the initial insult (Tables 1 and 2). Balazs et al. (1972) reported that adult Sherman rats pretreated with isoproterenol again became sensitive to isoproterenol challenge 11 weeks after the initial treatment. The longer duration of resistance observed in this study may be attributable to the strain or age of the animal or to various other undetermined factors. The data from Experiments II and III further show that acute lesion scores of isoproterenol- and saline-pretreated rats were not statistically different in either experiment when the animals were challenged with isoproterenol on Day 5 (Tables 2 and 3). It appears from these results that a 5-day period was not long enough for the development of myocardial resistance in rats. Bhatia et al. (1980) observed that rats which were pretreated with isoproterenol have more hypoxic tolerance 14 days after pretreatment than after 48 hr; they suggested that temporal delays are involved in the development of metabolic changes in the myocardium, which help in some way to develop resistance against the effects of hypoxia. The average lesion area occupied by the scar in rats pretreated for 10 consecutive days with isoproterenol was about 15% of the section area, while the corresponding value in rats with single-day pretreatment was only 4%. Although the area of the old lesions was significantly less in the latter case, no differences in the development of myocardial resistance was noted between the groups. As shown by these results, the resistance manifests itself even if the scar occupies only a small portion of the most sensitive subendocardial area, and thus the lack of responsiveness to isoproter-
enol challenge is not likely to be due to elimination of all susceptible areas by the previous insults. Lesions were present in rats treated with a single sc injection of isoproterenol at 5.0 pg/ kg but were absent or very minimal in rats that received 0.5 pg/kg. When the rats of both these groups were challenged with isoproterenol on Day 2 1, the group pretreated with 5.0 pg/kg had a significantly lower score of fresh lesions than the group that received 0.5 Fg/ kg, showing that rats which had initial lesions had some degree of resistance while those which did not have previous lesions did not manifest any resistance. These data support the hypothesis (Balazs, 1974) that the initial development of the lesion is a prerequisite for the resistance phenomenon. The mechanism of the resistance phenomenon is not understood. Since the cardiotoxic effect of isoproterenol is believed to be due to the exaggerated pharmacologic effect (Rona et al., 1963), it is reasonable to assume that the decreased sensitivity to isoproterenol challenge may be associated with increasing pharmacologic tolerance. Johnson et al. ( 1980) reported that the cardiovascular responses to isoproterenol and the number of receptor binding sites for [3H]dihydroalprenolo1 were not different in isoproterenol-pretreated and control rats. These findings suggest that the resistance phenomenon may not be due to alteration of the intensity of pharmacologic events. Cellular hypoxia is believed to play a major role in the production of myocardial necrosis induced by the sympathomimetic amines. An adaptation to hypoxia has been suggested as one of the probable mechanisms in the development of resistance. During hypoxia, the LDH isoenzyme pattern of cultured cells changes from predominantly heart (H) to muscle (M) subunits in both heart (Dawson et al., 1964) and liver (Johansson, 1966). Wenzel and Lyon (1967) demonstrated that isoproterenol pretreatment in rats produced a shift in the LDH isoenzyme pattern of the heart from H to M subunits and suggested
MYOCARDIAL
RESISTANCE
that increased formation of the LDH-M subunit may be a mechanism by which the heart adapts to an anaerobic environment. Moreover, Kaplan (1965) showed that fetal heart, which is relatively resistant to hypoxia, contains predominantly LDH-M while postnatal heart contains mostly LDH-H subunits. Bhatia et al. ( 1980) reported that the higher hypoxia tolerance exhibited by isoproterenoltreated rats can be eliminated by prior adrenalectomy but not by adrenal medullectomy. These authors postulated that the higher glycogen content of the heart brought about by increased blood levels of corticosteroids may be one of the factors responsible for the increased tolerance. An increase in anaerobic glycolysis has been detected histochemically in the area surrounding the myocardial infarct experimentally produced by coronary artery ligation in rats (Dusek et al., 197 1b), further demonstrating a morphological evidence of adaptation. Their data also showed that cardiac muscle cells of the adult mammalian myocardium can undergo considerable metabolic and structural adaptation, permitting survival under unfavorable conditions. Abri and Hecht (198 1) recently demonstrated that long-term application of small doses of isoproterenol produces an adaptive reaction in the heart muscle, resulting in an increased tolerance to ischemic injury. It is conceivable from all the above studies that the development of resistance is an adaptive phenomenon of the myocardium, most likely to the hypoxia-inducing effects of isoproterenol. ACKNOWLEDGMENT The authors extend their gratitude to Bonnie Markovitz for computer processing of the data.
205
TO ISOPROTERENOL
genesis and prevention of necrotizing cardiomyopathies. In Electrolytes and Cardiovascular Diseases (E. Bajusz, ed.), pp. 274-322. Karger, BaseI, New York. BALAZS, T. (I 974). Development of tissue resistance to toxic effects of chemicals. Toxicology 2, 247-255. BALAZS, T., OHTAKE, S., AND NOBLE, J. F. (1972). The development of resistance to the ischemic cardiopathic effect of isoproterenol. Toxicol. Appt. Pharmacol. 21, 200-213.
BHATIA, B., DATTA, K., AND NIJHAWAN, R. (1980). Hypoxic tolerance of rats with isoproterenol-induced myocardial necrosis. In Advances in Myocardiology (M. Tajuddin, B. Bhatia, H. H. Siddiqui, and G. Rona, eds.), vol. 2. pp. 153- 163. University Park Press, Baltimore. BLOOM, S. (198 1). Reversible and irreversible injury: Calcium as a major determinant. In Cardiac Toxicology (T. Balazs, ed.), vol. 1, pp. 179- 199. CRC Press, Boca Raton, Florida. DAWSON, D. M., GOODFRIEND, T. L., AND KAPLAN, N. 0. (1964). Lactic dehydrogenases: Functions of the two types. Science 143,929-933. DUSEK, J., RONA, G., AND KAHN, D. S. (1970). Myocardial resistance: A study of its development against toxic doses of isoproterenol. Arch. Pathol. 89, 79-82. DUSEK, J., RONA, G., AND KAHN, D. S. (1971a). Myocardial resistance to isoprenaline in rats: Variation with time. J. Pathol. 105, 279-282. DUSEK, J., RONA, G., AND KAHN, D. S. (197 1b). Healing process in the marginal zone of an experimental myocardial infarct. Amer. J. Pathol. 62, 321-332. HOYLAND, A. (1965). Robustness of the Hodges-Lehmann estimates for shift. Ann. Math. Stat. 36, 174197.
JOHANSSON,G. (1966). Influence of oxygen in the lactate dehydrogenase isozyme pattern in Chang liver cells. Exp.
Cell Res. 43, 95-97.
JOHNSON,G. L., BALAZS, T., EHRREICH,S. J., KENIMER, J. G., AND BLOOM, S. (1980). On the resistance to isoproterenol-induced cardiomyopathy in rats. Fed. Proc. Fed. Amer.
Sot. Exp. Biol.
39, 308.
KAPLAN, N. 0. (1965). Effect of hormones and environmental factors on lactic dehydrogenases. J. Cell. Comp. Physiol. 66, l-10. MARAMAA, S., AND Lnv, I. (1973). Zur resistenz gegenuber der kardionekrotischen wirkung des isoprenalins. Z. Kardiol.
62, 441-450.
RONA, G., KAHN, D. S., AND CHAPPEL, C. I. (1963). Studies on infarct-like myocardial necrosis produced by isoproterenol: A review. Rev. Can. Biol. 22, 241255.
REFERENCES ABRI, O., AND HECHT, A. (198 1). The influence of longterm application of isoprotemnol. Exp. Pathol. 20, 146152.
BAJUSZ, E. (1965). The terminal electrolyte shift mechanisms in heart muscle: Its significance in the patho-
SELYE, H., VEILLEUX, R., AND GRASSO, S. (1960). Protection by coronary ligature against isoproterenol-induced myocardial necrosis. Proc. Sot. Exp. Biol. Med. 104, 343-345.
WENZEL, D. G., AND LYON, J. P. (1967). Sympathomimetic amines and heart lactic dehydrogenase isoenzymes. Toxicol. Appl. Pharmacol. 11, 2 15-228.
AND APPLIED PHARMACOLOGY
Determinants
of Resistance
XAVIERJOSEPH,*
69, 199-205 (1983)
to the Cardiotoxicity
of lsoproterenol
SHERMAN BLOOM,? GORDONPLEDGER,*
*National Center for Drugs and Biologics, Food iDepartment of Pathology, George Washington
Received
November
in Rats
ANDTIBORBALAZS*
and Drug Administration, Washington, D.C. 20204 and University Medical Center, Washington, D.C. 20037
4. 1982: accepted
February
9, 1983
Determinants of Resistance to the Cardiotoxicity of Isoproterenol in Rats. JOSEPH, X., BLOOM, S., PLEDGER, G., AND BALAZS, T. (1983). Toxicol. Appl. Pharmacol. 69, 199-205. Induction of myocardial necrosis by isoproterenol produces resistance to the necrogenic effects of subsequent doses of the drug. A series of experiments were performed to further define the determinants of resistance. Myocardial necrosis was induced in male Sprague-Dawley rats by SCinjection of isoproterenol at 50 pglkg daily for 10 consecutive days or as a single dose at 50, 5, or 0.5 pg/ kg. These preconditioning doses were followed, at various times, by a challenge dose of 50 ccgl kg. The rats were killed 48 hr after the challenge dose, and their hearts were analyzed morphometrically to determine the amount of acute necrosis and scarring. The amount of scar tissue was a reflection of necrosis caused by the preconditioning dose whereas acute necrosis reflected response to the challenge dose. Resistance occurred and lasted longer than 19 to 20 weeks after both single or multiple isoproterenol injections of 50 ccg/kg,but it was not observed 5 days after administration of a single preconditioning dose.Isoproterenol at 0.5 pg/kg produced only very minimal or no myocardial necrosis and did not produce resistance. The resistance was not dependent on the size of the area of necrosis produced during the preconditioning period, showing that it was not due to destruction of all vulnerable muscle by the preconditioning dose(s). The preexistence of lesions, however, was necessary for the development of resistance. It is concluded that development of resistance to the necrogenic effects of isoproterenol reflects an adaptive alteration in the myocardium which survives after a necrogenic dose.
There are reports about the lack of sensitivity of rat myocardium to the necrosis-inducing effects of isoproterenol after an initial insult by either drugs (Balazs et al., 1972) or other means such as coronary artery ligation (Selye et al., 1960; Dusek et al., 1970) or strenuous muscular exercise (Bajusz, 1965). This phenomenon has usually been referred to as myocardial resistance or protection (Dusek et al., 197 la). Selye et al. (1960) showed that ligation of the left coronary artery in rats provided resistance against isoproterenol-induced myocardial necrosis when the drug was given a few days after ligation. Dusek et al. (1970) found that the protective effect was independent of the location of initial necrosis in the heart; they also reported that the myo-
cardial resistance was maximal 48 hr after coronary ligation and then gradually declined so that the myocardium was again sensitive to isoproterenol-induced necrosis 3 weeks after the ligation (Dusek et al., 1971a). Balazs et al. ( 1972) found that myocardial resistance to isoproterenol challenge occurred in rats pretreated with isoproterenol for 2 consecutive days and that the sensitivity to isoproterenol had returned 11 weeks after the initial treatment. Our present investigation was undertaken to study the development of resistance to isoproterenol challenge and its duration in rats which were pretreated with daily injections of isoproterenol for 10 consecutive days or with a single injection. In addition, experiments 199
0041-008X/83 Copyright All n&s
$3.00
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
200
JOSEPH ET AL.
were performed to determine whether the development of resistance is related to elimination of the most sensitive subendocardial area by the preceding insults and whether the initial development of myocardial lesions is a prerequisite for the resistance phenomenon.
Histopathology
In all three experiments, animals were killed by CO2 inhalation and were necropsied. Hearts were examined in situ, removed, rinsed in isotonic saline, and again examined for gross lesions. Serial transverse sections, 2-mm thick, were made from the apex to the base of the heart; these sections included both right and left ventricles. For microscopic studies, the second and fourth sections, counting from the apex, were fixed in 4% buffered formMETHODS aldehyde and processed by routine histologic procedures. Sections were cut at a normal thickness of 5 Mm and Male Sprague-Dawley rats weighing 400 to 500 g (3 stained by Masson’s trichrome method; they were asto 4 months old) were obtained from Charles River Breedsigned random numbers to ensure unbiased analysis. ing Laboratories, Wilmington, Mass. and were housed Morphometric analysis was performed by projecting the image of the slide onto graph paper and counting the two in a cage with free accessto feed and water. frequency with which normal or abnormal areas of the myocardium overlapped points of intersection on the paExperiment I per. The apical-most section of ventricular myocardium was used for grading. Three morphological features were used for evaluation purposes: (1) normal tissue, which One hundred forty rats were randomly assigned to two stained red, (2) acutely necrotic tissue with inflammatory equal groups. Rats of the first group were given DL-iSOproterenol HCl (Sigma Chemical Co., St. Louis, MO.) at cells, edema fluid, and necrotic muscle fibers, which stained 50 &kg sc for 10 consecutive days (Days 1 to 10) and light blue; and (3) scars with dense collagen, which stained rats of the second group received saline. On each of Days dark blue. The percentage of the area represented by each 9 and 10, 10 rats from the second group were treated SC of these features was calculated. with isoproterenol at 50 pg./kg, and these rats and 10 rats from the first group were killed on Day 11. Ten rats that had received only saline were also killed on the same day. Statistical Analysis Of the remaining rats, 10 from each group were challenged Results were analyzed statistically by Wilcoxon onewith isoproterenol at 50 &kg sc for 2 days beginning on Days 2 1, 37, 7 1, or 14 1 and were killed 24 hr after the sample test procedures. Values are expressed as sample median lesion scores (SMLS) with 95% confidence intersecond challenge dose. vals for the median, unless otherwise indicated. (These values are actually pseudomedians as defined by Hoyland (1965), but are very close to median values for data of Experiment II this nature.) Each group was compared with its concurrent control group for significance by Wilcoxon rank-sum Seventy rats were given a single sc injection of isoproterenol HCI at 50 &kg (Day 1) and another group of 70 tests. The differences were considered significant at p < 0.05. rats received saline. Eight rats from each group were killed 48 hr later. On Days 5, 12, 21, 43, 82, and 134, 8 to 10 rats each from both the isoproterenol- and saline-treated RESULTS groups were challenged with a single SCinjection of isoproterenol (50 &kg) and were killed 48 hr later.
Experiment Experiment Ill Seventy-two rats were assigned to three equal groups. Rats of the first and second groups were given a single sc injection of isoproterenol HCI at 5.0 and 0.5 &kg, respectively, and rats of the third group were given saline. Eight animals from each of the three groups were killed 48 hr later. Of the remaining rats, eight from each group were challenged with a single injection of isoproterenol (50 &kg sc) on Days 5 or 21 and were killed 48 hr thereafter.
I
Twelve of the seventy rats treated with isoproterenol for 10 consecutive days died during the treatment period. A total of ten rats from this pretreated group also died at various intervals during isoproterenol challenge. From the control group pretreated with saline and then challenged with isoproterenol, a total of 20 rats died during different intervals. Gross examination of the hearts of rats that
MYOCARDIAL
RESISTANCE
were treated with isoproterenol for 10 days revealed marked pale greyish areas of lesions at the apical region of the ventricular walls. Gross heart lesions were also observed in most of the animals that had received only 2 days of treatment with isoproterenol. Histologically, the animals treated for 2 days showed fresh myocardial necrosis, and those treated for 10 days showed lesions in various stages of healing, including well-healed scars. The isoproterenol-induced lesions have been previously described in detail (Bloom, 1981). The lesions were mainly seen at the apex, left ventricular walls, and papillary muscles; most were localized in the subendocardial layer of the free wall and septum of the left ventricle. Fresh lesions consisted of small foci or multiple areas of muscle necrosis associated with interstitial edema and inflammatory cell infiltration. The inflammatory infiltrate consisted mainly of mononuclear cells and rarely of neutrophils. The necrotic muscle cells
TO ISOPROTERENOL
201
showed a loss of cross striations and the presence of prominent hypercontraction bands. The right ventricular wall was not usually involved. Healing lesions consisted of varying amounts of collagen and fibroblasts with or without chronic inflammatory cells, depending on the age of the lesion. Old lesions consisted of dense scars, with no inflammatory cells and a few fibroblasts. Some of these changes are shown in Fig. 1. In rats that received both the initial or preconditioning dose and a subsequent challenge dose, the scar represented necrosis caused by the preconditioning dose and the acute necrosis, if any, reflected response to the challenge dose. Table 1 summarizes the extent of myocardial lesions in rats pretreated with isoproterenol or saline consecutively for 10 days and challenged at different intervals. The amount of fresh myocardial necrosis after 10 days of treatment with isoproterenol was minimal (SMLS 0.3 compared with 11.4 for acute con-
FIG. 1. Photomicrograph of left ventricular myocardium of rat given 50 pg isoproterenol HCl/kg once daily for 10 days, challenged with the same dose on days 141 and 142, and killed on day 143. Dense scar (a) is present, as well as acute necrosis (b) and normal muscle (c). Masson’s trichrome stain, 300X.
202
JOSEPH ET AL. TABLE 1 EXTENT
OF MY~CARDIAL FOR
LESIONS
10 CONSECUTIVE
IN RATS
DAYS
AND
PRETREATED CHALLENGED
WITH
ISOPROTERENOL
WITH
IS0
AT VARIOUS
HCI
(ISO)
OR SALINE
INTERVALS
Sample median lesion scores (%) with 95% confidence interval” Days of challenge (ISO 50 a/kid
Day killed
IS0 pretreatment”
Saline pretreatmenth
Acute necrosis
Scar
Acute necrosis
No challenge 9, 10 21,22 37, 38 71, 72 141, 142
11 11 23 39 73 143
0.3 (O-6.2) 0 (O-0.2) 0.8 (O-4.2) 1.6 (O-3.4) 3.7 (2.0-4.6)
16. I (9.4-22.5) 18.6 (8.2-34.8) 10.9 (6.3-17.3) 16.1 (10.9-22.0) 15.6 (5.9-25.1)
0.3 (O-0.7) 11.4 (6.6-16.9) 14.0 (6.0-22.3)’ 11.2 (5.6-19.1)’ 8.4 (2.1-23.1)’ 20.2 (6.8-34.2)‘:d
Scar 0.3 0 0 0.2 0 0.5
(O- 1.1) (O-0.2) (O-0.1)’ (O-0.4)’ (O-1.7)’ (0.3-I.o)c”
’ Each value represents the sample median lesion score from 5 to 10 hearts. ’ Pretreatment with IS0 at 50 wg/kg SCor saline was given for 10 consecutive days (Days 1 to 10). ’ Significantly different from value of concurrent ISO-pretreated group (p i 0.05). d 88% confidence interval.
trols), and the amount of old lesions was extensive (SMLS 16.1 compared with zero for acute controls). The saline-treated rats usually did not have any lesions. The amount of acute necrosis in pretreated animals was significantly less than that in their concurrent controls at each of the four challenge periods, i.e., on Days 21, 37, 71, and 141. The amount of scarring was significantly greater in isoproterenol-pretreated groups at each stage, compared with their respective controls. Even though there appeared to be a trend for increased fresh myocardial necrosis in the isoproterenol-pretreated group with passage of time, i.e., from Days 2 1 to 14 1, the values for the respective control groups were always significantly higher. Experiment
II
In Experiment II, 1 of 70 rats died on the first day of isoproterenol treatment, and a total of 5 from the pretreated group died during isoproterenol challenge at various intervals. From the saline-treated group, only two rats died during isoproterenol challenge. The extent of the myocardial lesions in various groups of rats is summarized in Table 2. The
lesions were less severe than those occurring in the first experiment. Rats that were pretreated with a single SCinjection of isoproterenol and challenged on Days 12, 21, 82, or 134 had significantly lower scores of acute myocardial lesions (p < 0.05) compared with their concurrent controls pretreated with saline and challenged with isoproterenol. The amount of scarring was significantly greater in the above groups than in their respective controls. The fresh and old lesion scores in rats pretreated with isoproterenol and challenged on Days 5 and 43 were not significantly different from the control values. Experiment
III
All the rats in Experiment III survived. Table 3 summarizes the extent of myocardial lesions in rats pretreated with a single SCinjection of low doses of isoproterenol (5.0 or 0.5 pg/kg) or saline and then challenged with doses 10 to 100 times higher. Single SCinjections of isoproterenol at 5.0 pg/kg produced significantly more acute lesions (SMLS 1.2) compared with the group that received 0.5 cLg/ kg (SMLS 0.1). Saline-treated rats had no lesions. The acute lesion scores of rats that were
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RESISTANCE
203
TO ISOPROTERENOL
TABLE 2 EXTENT
OF MY~CARDIAL
LESIONS IN RATS PRETREATED WITH A SINGLE DOSE OF LWPROTERENOL OR SALINE AND CHALLENGED WITH IS0 AT VARIOUS INTERVALS
HCl (ISO)
Sample median lesion scores (a) with 95% confidence interval’ Day of challenge (ISO 50 /.&kg)
Day killed
IS0 pretreatment * Acute necrosis
No challenge 5 12 21 43 82 134
3 7 14 23 45 84 136
6.0 (3.2-10.0) 1.3 (0.8-2.8) 0.7 (0.2-1.9) 0.6 (0.1-1.5) 2.4 (1.5-3.9) 0.9 (0.2-l .8) 0.2 (0-2.4)d
Saline pretreatment*
Scar 0.5 4.0 3.8 5.0 2.7 3.0 4.1
Acute necrosis
(0.1-0.8) (0.2-13.1) (2.4-7.6) (2.0-8.2) (0.8-5.6) (1.7-8.9) (2.7-6.4)d
0.4 3.2 4.0 5.4 4.2 6.3 5.4
(O-2.7) (0.5-5.8) (2.7-10.8)’ (1.8-8.8)C (2.0-12.4) (1.1-13.2)’ (1.4-8.1)’
scar 0.5 (0.2-0.8) 1.0 (0.5-1.6) 0.3 (0. I-O.S)C 0.8 (0.3-1.6)’ 0.8 (0.4-l .2) 1.5 (0.7-2.4)’ 1.8 (o-4.0)’
’ Each value represents the sample median lesion score from 5 to 10 hearts. ’ Pretreatment with a single sc dose of IS0 at 50 &kg or saline was given on Day 1. ’ Significantly different from value for concurrent ISO-pretreated group (p < 0.05). d 98% confidence interval.
pretreated with a single SC injection of isoproterenol at 5.0 or 0.5 &kg and challenged with a single dose at 50 pg/kg on Day 5 were not significantly different from the control values. The acute lesion score in rats pretreated with isoproterenol at 5.0 &kg and challenged with 50 bg/kg on Day 21 was significantly less (SMLS 1.5) compared with rats pretreated with 0.5 pg/kg and challenged with 50 pg/kg (SMLS 7.5). Even though the acute
lesion score was much lower in the group pretreated with 5.0 pg/kg (SMLS 1.5) than in the saline-pretreated group (SMLS 4.7), the values were not statistically different. DISCUSSION The results of the present study indicate that pretreatment with isoproterenol decreases the sensitivity of rat myocardium to
TABLE 3 EXTENT OF MY~CARDIAL LESIONS IN RATS PRETREATED WITH Low DOSES OF ISOPROTERENOL OR SALINE AND CHALLENGED WITH IS0 AT VARIOUS INTERVALS
HCl (ISO)
Sample median lesion scores (%) with 95% confidence interval” IS0 pretreatment
b
5.0 a/k Day of challenge (ISO 50 fig/kg)
Day killed
No challenge 5 21
3 7 23
Acute necrosis 1.2 (0.149) 2.3 (1.1-8.7) 1.5 (0.3-4.0)
Saline pretreatmentb
0.5 e/kg Scar
0.7 (0.4-1.2) 1.6 (0.8-2.5) 1.0 (0.7-1.4)
Acute necrosis 0.1 (O-1.0)’ 3.9 (1.6-10.9) 7.5 (1.8-13.4)’
SCX 0.6 (0.3-1.0) 0.6 (0.2-1.4) 0.7 (0.4-0.9)
a Each value represents the sample median lesion score from 6 to 10 rats. b Pretreatment with a single sc dose of IS0 at 5.0 or 0.5 &kg or saline was given on Day 1. ’ Significantly different from value for rats pretreated with 5.0 &kg ISO.
Acute necrosis 0 5.7 (1.4-10.5) 4.7 (1.0-14.9)
scar 0.9 (0.4-1.8) 0.7 (O-2.2) 1.1 (0.7-2.9)
204
JOSEPH ET AL.
the lesion-inducing effects of a subsequent challenge; i.e., after an initial injury, the myocardium develops a resistance to further lesion-inducing insults. This finding is consistent with the findings of other investigators (Balazs et al., 1972; Maramaa and Liiv, 1973). The data also show that the sensitivity to isoproterenol of rats pretreated with either single or multiple doses of isoproterenol does not return even 19 to 20 weeks after the initial insult (Tables 1 and 2). Balazs et al. (1972) reported that adult Sherman rats pretreated with isoproterenol again became sensitive to isoproterenol challenge 11 weeks after the initial treatment. The longer duration of resistance observed in this study may be attributable to the strain or age of the animal or to various other undetermined factors. The data from Experiments II and III further show that acute lesion scores of isoproterenol- and saline-pretreated rats were not statistically different in either experiment when the animals were challenged with isoproterenol on Day 5 (Tables 2 and 3). It appears from these results that a 5-day period was not long enough for the development of myocardial resistance in rats. Bhatia et al. (1980) observed that rats which were pretreated with isoproterenol have more hypoxic tolerance 14 days after pretreatment than after 48 hr; they suggested that temporal delays are involved in the development of metabolic changes in the myocardium, which help in some way to develop resistance against the effects of hypoxia. The average lesion area occupied by the scar in rats pretreated for 10 consecutive days with isoproterenol was about 15% of the section area, while the corresponding value in rats with single-day pretreatment was only 4%. Although the area of the old lesions was significantly less in the latter case, no differences in the development of myocardial resistance was noted between the groups. As shown by these results, the resistance manifests itself even if the scar occupies only a small portion of the most sensitive subendocardial area, and thus the lack of responsiveness to isoproter-
enol challenge is not likely to be due to elimination of all susceptible areas by the previous insults. Lesions were present in rats treated with a single sc injection of isoproterenol at 5.0 pg/ kg but were absent or very minimal in rats that received 0.5 pg/kg. When the rats of both these groups were challenged with isoproterenol on Day 2 1, the group pretreated with 5.0 pg/kg had a significantly lower score of fresh lesions than the group that received 0.5 Fg/ kg, showing that rats which had initial lesions had some degree of resistance while those which did not have previous lesions did not manifest any resistance. These data support the hypothesis (Balazs, 1974) that the initial development of the lesion is a prerequisite for the resistance phenomenon. The mechanism of the resistance phenomenon is not understood. Since the cardiotoxic effect of isoproterenol is believed to be due to the exaggerated pharmacologic effect (Rona et al., 1963), it is reasonable to assume that the decreased sensitivity to isoproterenol challenge may be associated with increasing pharmacologic tolerance. Johnson et al. ( 1980) reported that the cardiovascular responses to isoproterenol and the number of receptor binding sites for [3H]dihydroalprenolo1 were not different in isoproterenol-pretreated and control rats. These findings suggest that the resistance phenomenon may not be due to alteration of the intensity of pharmacologic events. Cellular hypoxia is believed to play a major role in the production of myocardial necrosis induced by the sympathomimetic amines. An adaptation to hypoxia has been suggested as one of the probable mechanisms in the development of resistance. During hypoxia, the LDH isoenzyme pattern of cultured cells changes from predominantly heart (H) to muscle (M) subunits in both heart (Dawson et al., 1964) and liver (Johansson, 1966). Wenzel and Lyon (1967) demonstrated that isoproterenol pretreatment in rats produced a shift in the LDH isoenzyme pattern of the heart from H to M subunits and suggested
MYOCARDIAL
RESISTANCE
that increased formation of the LDH-M subunit may be a mechanism by which the heart adapts to an anaerobic environment. Moreover, Kaplan (1965) showed that fetal heart, which is relatively resistant to hypoxia, contains predominantly LDH-M while postnatal heart contains mostly LDH-H subunits. Bhatia et al. ( 1980) reported that the higher hypoxia tolerance exhibited by isoproterenoltreated rats can be eliminated by prior adrenalectomy but not by adrenal medullectomy. These authors postulated that the higher glycogen content of the heart brought about by increased blood levels of corticosteroids may be one of the factors responsible for the increased tolerance. An increase in anaerobic glycolysis has been detected histochemically in the area surrounding the myocardial infarct experimentally produced by coronary artery ligation in rats (Dusek et al., 197 1b), further demonstrating a morphological evidence of adaptation. Their data also showed that cardiac muscle cells of the adult mammalian myocardium can undergo considerable metabolic and structural adaptation, permitting survival under unfavorable conditions. Abri and Hecht (198 1) recently demonstrated that long-term application of small doses of isoproterenol produces an adaptive reaction in the heart muscle, resulting in an increased tolerance to ischemic injury. It is conceivable from all the above studies that the development of resistance is an adaptive phenomenon of the myocardium, most likely to the hypoxia-inducing effects of isoproterenol. ACKNOWLEDGMENT The authors extend their gratitude to Bonnie Markovitz for computer processing of the data.
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genesis and prevention of necrotizing cardiomyopathies. In Electrolytes and Cardiovascular Diseases (E. Bajusz, ed.), pp. 274-322. Karger, BaseI, New York. BALAZS, T. (I 974). Development of tissue resistance to toxic effects of chemicals. Toxicology 2, 247-255. BALAZS, T., OHTAKE, S., AND NOBLE, J. F. (1972). The development of resistance to the ischemic cardiopathic effect of isoproterenol. Toxicol. Appt. Pharmacol. 21, 200-213.
BHATIA, B., DATTA, K., AND NIJHAWAN, R. (1980). Hypoxic tolerance of rats with isoproterenol-induced myocardial necrosis. In Advances in Myocardiology (M. Tajuddin, B. Bhatia, H. H. Siddiqui, and G. Rona, eds.), vol. 2. pp. 153- 163. University Park Press, Baltimore. BLOOM, S. (198 1). Reversible and irreversible injury: Calcium as a major determinant. In Cardiac Toxicology (T. Balazs, ed.), vol. 1, pp. 179- 199. CRC Press, Boca Raton, Florida. DAWSON, D. M., GOODFRIEND, T. L., AND KAPLAN, N. 0. (1964). Lactic dehydrogenases: Functions of the two types. Science 143,929-933. DUSEK, J., RONA, G., AND KAHN, D. S. (1970). Myocardial resistance: A study of its development against toxic doses of isoproterenol. Arch. Pathol. 89, 79-82. DUSEK, J., RONA, G., AND KAHN, D. S. (1971a). Myocardial resistance to isoprenaline in rats: Variation with time. J. Pathol. 105, 279-282. DUSEK, J., RONA, G., AND KAHN, D. S. (197 1b). Healing process in the marginal zone of an experimental myocardial infarct. Amer. J. Pathol. 62, 321-332. HOYLAND, A. (1965). Robustness of the Hodges-Lehmann estimates for shift. Ann. Math. Stat. 36, 174197.
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