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Myocardial injury following endogenous catecholamine release in rabbits
j Mol Cell Cardio117, 377-387 (1985)
Myocardial Injury Following Endogenous Catecholamine R e l e a s e in Rabbits S. Evans Downing and Victor Chen Department of Pathology, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06510, USA (Received I June 1984, accepted in revisedform 7 September 1984) S. EvAns Dow~I~G AND V. CIaEN. Myocardial Injury Following Endogenous Catecholamine Release in Rabbits. Journal of Molecular and Cellular Cardiology (1985) 17, 377 387. Catecholamines (CAT) given in large doses produce cardiomyopathic changes in several animal species. This study was designed to determine if endogenous release can also induce cardiac injury, Rabbits were infused with doses of tyramine (TYR), ranging from 200 to 500 #g/min/kg, i.v. for 90 rain. Arterial pressure and heart rate were measured, as were total C A T concentrations, blood gases, pH and glucose. Two days later the animals were killed and cardiac injury assessed using a histological scoring system. All data were compared with controls given saline. Initial C A T averaged 452 pg/ml, rose to 2890 pg/ml after starting TYR, 500 ktg/min/kg, and remained elevated for the duration of infusion. Circulating C A T levels were a function of T Y R dose, and bore a linear relationship to the histological score (P < 0.001). Development of lesions was unaltered by betaj blockade with practolol, but sharply reduced by alpha blockade with phentolamine (P < 0.01). Pretreatment with insulin also reduced lesion formation, but diabetic (alloxan) rabbits showed no greater C A T injury. It is concluded that endogenous release of C A T induces myocardial injury in the rabbit in a dose-dependent manner. This is unrelated to myocardial O 2 demand, and microvascular pathology was absent. Activation of alpha adrenergic pathways is likely the dominant or exclusive mechanisml KEY WORDS: Cardiomyopathy; Tyramine; Catecholamines; Rabbits; Practolol; Phentolanfine; Alloxan diabetes ; Insulin ; Cardiac O2demand ; Cardiac muscle necrosis.
Introduction wtlich provides a consistent and reproducible Exogenous administration of large doses of cardiomyopathy with limited quantitative catecholamines is known to cause myocardial variability as judged by a histological scoring damage in several animal species [4, 11, 13, 15, system [4]. Using a constant time base of 90 23, 24, 27, 29]. Similar patterns of cardiac rain, infusions of relatively modest doses of NE injury have been described in humans as well (2 to 3 /2g/min/kg) is sufficient to produce [10, 23, 29]. Several adrenergic agonists have extensive myofiber injury judged by light been employed in experimental studies, and microscopy and also by radionuclide cardiac these have yielded similar results. Thus nor- imaging [22]. Moreover, the magnitude of epinephrine [4, 5, 11, 13, 23], epinephrine [11, cardiac injury is dose-related [5]. Physiologi23] and isoproterenol [1, 9, 24] have all been cal studies have demonstrated substantial found to cause myofiber injury with appar- impairment of left ventricular performance in ently identical histological patterns. While this model [17, 32]. this serves as a common denominator, interWhereas previous work establishes that the pretation of the findings and possible patho- catecholamines given in large doses leads to genetic mechanisms have been confounded by important cardiomyopathic changes, it is less widely varying protocols, and doses which are clear whether endogenous catecholaminc release is potentially injurious. The exception often massive [-1]. Based on earlier studies by Schenk and is data from human subjects with malignant Moss [27], we have developed a rabbit model pheochromocytoma [14, 29, 30, 31]. A limited 0022-2828/85/040377 + 11 $03.00/0
9 1985 Academic Press Inc. (London) Limited
378
S. Evans D o w n i n g and V. Chen
number of these patients exhibited lesions similar to those found in experimental catecholamine injury. However, these are rare cases and do not address the question of potential myofiber injury by circulating catecholamines released endogenously in the normal organism. The objectives of the present study were to determine in the rabbit model if endogenous release of catecholamines using tyramine can elicit myocardial damage ; to establish dose-response relations; to measure changes in circulating catecholamines and their relationship to lesion formation; and to determine the receptors primarily responsible by using adrenoceptor blocking agents. The question of enhanced sensitivity to myofiber damage in the insulin deficient state [4] was also explored in animals with alloxan diabetes.
phentolamine (Ciba-Geigy), 2.5 mg/kg. Insulin (10 U/kg) was given to five animals 30 min before tyramine infusion was begun. Effects of insulin deficient diabetes mellitus on tyramine-induced myocardial injury was studied in ten animals given altoxan monohydrate (Sigma), 100 to 110 mg/kg, as an i.v. bolus injection. Tyramine infusions were performed 10 to 40 days after alloxan treatment. Arterial blood samples were obtained before, and after 5, 15, 60 and 90 min oftyramine infusion to determine circulating catecholamine concentrations. Total plasma catecholamines were measured with a commercially available kit (Cat-A-Kit, Upjohn Diagnostics). The radioenzymatic assay involves the transfer of a [3H]methyl group from S-adenosyl-L-methionine-[3H]methyl to epinephrine or norepinephrine which is catalyzed by the enzyme catechol-o-methyltransferase. The resulting [3H]methoxy Methods derivatives are oxidized to [3H]vanillin, Data to be presented in this study were extracted and quantified by liquid scintilobtained from 74 New Zealand white rabbits lation counting. of either sex, and with a mean body weight of Following infusion the catheters were 4.0 kg. All animals were anesthetized with removed, the femoral wound surgically closed pentobarbital, 30 mg/kg and polyethylene and the animals returned to their cages after catheters were placed in a femoral artery and recovery from anesthesia. They were fed a vein. Arterial pressure was continuously mea- standard diet and water ad libitum. All animals sured with a Sanborn transducer and heart were killed 2 days later by an overdose of rate determined with a Sanborn cardio- pentobarbital via an ear vein. The hearts were tachometer. The latter was verified by immediately removed, emptied and weighed. manual assessment of pulse frequency from The atria and R V free wall were dissected and pressure traces inscribed by a Sanborn weighed, and the LV ( + s e p t u m ) separately (Model 7700) recorder. Body temperature weighed. Transverse 'ring' sections of LV was maintained with a heating pad. Arterial were obtained from the basal and mid porblood samples were obtained at 15 min inter- tions and fixed in 10% buffered formalin. vals and analyzed for Po2, Pco2 and p H They were prepared by standard histological using an Instrumentation Laboratories methods and stained with hematoxylin and analyzer. Hematocrit and glucose concentra- eosin for subsequent analysis. tions (Glucostat, Worthington Biochemical) Morphological evaluation employed a were also determined. semi-quantitative histological scoring system Tyramine (Sigma) was freshly prepared by described previously [4]. In brief, each section dilution in normal saline to concentrations was graded by two observers according to the that would provide the desired dose when extent and intensity of the leukocytic infused at a constant rate of 0.382 ml/min. response, without prior knowledge of the proDoses of 500, 400, 300 or 200 #g/min/kg (free cedures used in a given animal. A maximum base) were given i.v. for 90 min. Control score of 2.0 was given when the lesions were animals were similarly infused with 0.9% florid, extensive and transmural. Those with saline. In selected experiments adrenoceptor definite but sparse lesions were scored 1.0. blocking agents were given i.v. 10 rain prior to Equivocal focal lesions were scored 0.5. Those initiation of tyramine infusion. These judged to manifest injury more extensive than included practolol (Ayerst), 4 mg/kg; and 1.0, but less than 2.0 (e.g. nontransmural)
Cardiac Injury f r o m E n d o g e n o u s C a t e c h o l a m i n e s
were assigned a score of 1.5. A score of 0 was given when no histologic abnormality was present. Values for each of the two sections were averaged and used in scoring a given heart. Substantial alterations in arterial pressure and heart rate occurred during tyramine administration. Maximal changes and integrated mean values were determined from data obtained at 10 rain intervals. The pressure-rate product was calculated as an index of metabolic demand [25, 26]. All data were analyzed by standard statistical methods using the non-paired Student t-test and presented as mean values • of the mean. For multiple comparisons, one-way analysis of variance followed by the Scheffb test was employed [33]. Differences were considered significant when P was < 0.05.
Results
Circulatory responses to tyramine Average changes in arterial pressure, heart rate and the pressure-rate product measured in animals given tyramine, 500 #g/min/kg, are ~
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FIGURE l. Changes in arterial pressure, heart rate and pressure-rate product (P x R) during infusion of tyramine, 500 ( 0 - - 0 ) ,ug/min/kg (closed circles, n = 5) or normal saline (O--O) (open circles, n = 5). Vertical brackets, S.E.M.
379
shown in Figure 1. Mean arterial pressure (MAP) rose from 104 (__+2.6) m m H g to 148 (___9.5) m m H g within 2 rain. After 10 min it began to decline, and at 30 min had fallen to 115 (-t-7) m m H g , not significantly greater than initial control values. At 90 min, MAP was 86 (+2.1) m m H g , significantly lower than control values. In contrast, heart rate increased from 252 ( + 13) beats/rain to 330 ( + 1 1 ) at 10 min, and remained elevated throughout the duration of tyramine infusion. Saline infused animals showed no significant hemodynamic changes (Fig. 1, open circles). The pressure-rate (P x R) product (lower panel) increased substantially with tyramine infusion from 26.1 (-t-1.4) to 48.7 (-t-1.9) units and remained elevated throughout most of the infusion period. The mean P x R for the tyramine group was 34.8 (_+ 3.1), compared with the saline group which averaged 25.2 (_+ 0.6) (P < 0.05). Thus, maximal and average myocardial 0 2 requirements were likely substantially greater in the animals given tyramine. Metabolic measurements obtained for each group are listed in Table 1.
Morphological patterns of i n jury T h e characteristic pattern of myocardial injury resulting from infusion of larger doses of tyramine is illustrated in Figure 2. A heavy cellular infiltrate of predominantly mononuclear cells was uniformly present. Large histiocytic cells were most numerous, accompanied by less frequent lymphocytes. Granulocytes, including eosinophils, were occasionally present. The infiltrate was interstitial, and tended to concentrate in association with foci of myofiber necrosis. In addition to fragmentation and focal myofiber destruction, numerous contraction bands and zones of granularity consistent with swollen mitochondria were also evident with light microscopy. Z-lines were generally indistinct and myofiber nuclei were often lost in the more active inflammatory foci. These changes were in general most pronounced in the papillary muscles and inner half of the ventricular wall. However, a transmural distribution was often observed in hearts subjected to higher doses of tyramine. There was no distinction in the intensity of free wall or septal involvement. Neither the larger coronary arteries nor myocardial arterioles exhibited discernible
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TABLE 1. Arterial pH, Po2, Pco2, hematocrit and glucose concentrations
pH
Po2-TorI
Pco2-Torr
Hematocrit (%~
Glucose (mg/dl)
Time (min)
Saline
Tyramine-200
Tyramine-300
Tyramine-500
0 30 60 90 100 0 30 60 90 100 0 30 60 90 I00 0 30 60 90 100 0 30 60 90 100
7.37 + 0.02 7.44 +_ 0.03 7.47 __ 0.03 7.48 ___0.03 7.48 • 0.02 62.7+ 5.7 73.0 __+5.6 78.3 • 5.9 79.3 • 9.6 79.0 • 6.0 39.3 _ 2.9 33.7 • 2.5 30.7 _+ 1.8 29.0 • 1.9 29.7 • 3.2 38.0 • 1.9 36.3 • 2.9 36.7 + 2.0 35.3 ___2.3 35.0 • 1.9 147 +_ 11 157 _ II 157 • 11 160 • t4 153 • 15
7.42 _+ 0.02 7.48 + 0.04 7.50 + 0.03 7.51 • (1.03 7.50 • 0.02 68.4 • 4.3 72.4 • 4.2 80.2 + 3.1 83.8 • 1.6 88.6 ___ 1.8 40.2 • 1.3 32.6 • 1.4 30.2 • 1.1 28.4 + 2.0 24.8 • 0.8 43.6 • 1.0 41.6 • t.3 42.8 • 1.2 41.6 • 0.6 38.4 ___0.6 158 -t- 14 168 • 17 170 + 22 176 • 22 188 • 25
7.41 _+ 0.02 7.52 + 0.03 7.56 +__0.02 7.55 __+0.01 7.51 +_ 0.03 56.2 __+3.4 64.4 • 3.0 73.2 __ 4.7 82.8 __ 4.9 86.8 • 5.1 43.6 • 3.5 34.6 -t- 2.9 31.6 __+2.6 28.0 • 2.5 24.6 +_ 2.8 40.8 __+0.8 41.2 • 1.0 42.4 • 1.0 41,5 • 1.7 39,0 • 1.3 166 • 19 200 + 20 194 • 17 174• 21 224 _ 18
7.43 +_ 0.01 7.48 • 0.08 7.53 _+ 0.08 7.51 +__0.07 7.51 + 0.07 63.2 ___5.6 51.7 + 3.5 57.8 __ 3.5 50.5 __ 12.3 58.2 • 14.4 40.0 _ 1.5 37.3 _ 1.1 35.7 • 2.1 31.2 __+3.3 27.5 + 3.6 41.5 _ 0.9 40.3 • 1.9 42.0 _ 4.4 41.2 • 4.3 38.0 • 5.1 140 --4-9 225 • 16 235 • 20 202 +_ 28 230 + 33
pg/ml (n = 17). Five minutes after b e g i n n i n g tyramine, 200/~g/min/kg, the average concentration rose to 1573 (___373) pg/ml. This m a y be contrasted with t y r a m i n e given at the rate of 500 #g/min/kg. I n this group the increase after 5 m i n was nearly twice as great {2889 _ 209 pg/ml). After 15 min total catecholamine concentrations declined somewhat (Fig. 4), b u t in all groups values remained significantly higher t h a n saline controls for the entire 90 m i n infusion period. A highly significant relationship was found between the histological score applied to each heart a n d average catecholamine concentrations. These data are shown in Figure 5. Linear regression analysis demonstrated a correlation coefficient of 0.842 ~P < 0.001 ~. Circulating catecholamine concentrations Integrated mean values (averaged over 90 during tyramine administration rain) for total catecholamine concentrations Effects of infusing various concentrations of in excess of 1000 pg/ml resulted in significant t y r a m i n e on total circulating catecholamine myofiber damage. Concentrations of approxlevels are shown in Figure 4. Initial m e a n imately 2000 pg/ml were associated with values in each group were less t h a n 500 pg/ml, m a x i m a l myocardial injury as j u d g e d by the a n d concentrations averaged 451.5 (__+_26] histological scoring system.
histopathological changes. T h r o m b i were never e n c o u n t e r e d in 150 sections examined. T h e m e a n histological scores o b t a i n e d from 38 rabbits infused with various a m o u n t s of t y r a m i n e are shown i n Figure 3, and illustrate the dose response relationship. Scores ranged from 1.84 (__+0.06) in a n i m a l s given 500 # g / m i n / k g to 0.97 (-t-0.17) in those gxven 200 # g / k g / m i n ~P < 0.001 ). Eight animals infused with saline showed no definite lesions ( m e a n score, 0.16 ___0.10). Regression analysis revealed a correlation coefficient of 0.848 ( P < 0 . 0 0 1 1 . Analysis of variance d e m o n strated that the group means differ (P < 0.05).
Cardiac Injury from Endogenous Catecholamines
381
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FIGURE 2. (a). Photomicrograph of a typical histological section of left ventricular myocardium from a rabbit sacrificed 2 days after giving tyramine, 400 pg/min/kg for 90 min. H + E stain. Original magnification x 120. Intense interstitial cellular infiltrate, and myofiber separation and lossis evident. (b). Higher magnification (x 300). Myofiber vacuolization, granularity and degeneration is marked. The infiltrate is mononuclear, and consists mainly of cells with morphological features of histiocytes. Granulocytes are absent. This section was assigned a histological score of 2.0.
Effects of specific adrenoceptor blocking agents T o confirm that myofiber injury following t y r a m i n e was r e l a t a b l e to elevations in circulating catecholamines a n d receptor binding, selective blocking agents were employed. Results from 20 animals are presented in Figure 6 a n d T a b l e 2. T h e m e a n histological score from five rabbits given t y r a m i n e only (Ts00) was 1.80 (__+0.09). A n a d d i t i o n a l five
animals were p r e t r e a t e d with large doses (4 mg/kg) o f the beta I blocking agent, practolol ( + P 4 ) , p r i o r to initiation o f the t y r a m i n e infusion. T h e m e a n histological score of this g r o u p was 1.50 ( + 0.18), i n d i c a t i n g a persistent high level o f m y o c a r d i a l injury that d i d not differ from the group w i t h o u t beta blockade. This m a y be contrasted with animals p r e t r e a t e d with p h e n t o l a m i n e ( q - R i 0 ) in which the m e a n score was reduced to 0.53
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(-t-0.28). This difference was significant (P < 0.01) when compared with either of the preceding groups and indicates minimal or questionable injury. Similarly, in the five animals pretreated with both practolol and phentolamine ( + P 4 + Rt0) no significant lesions were identified, and the mean score was 0.05 ( + 0.02). This group differed significantly from the first two (P < 0.001), but not from the third group given phentolamine alone (-t-R10 ). These data indicate that myocardial injury following tyramine is largely or entirely relatable to activation of alpha adrenoceptor pathways, with little or no contribution from beta receptor activation. Circulatory changes summarized in Table 2 demonstrate that both blocking agents
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FIGURE 5. Relationship of average catecholamine concentrations during tyramine infusion period (90 rain) to histological scores of hearts obtained at sacrifice 2 days later. Data include 70 catecholamine measurements in 18 rabbits. Vertical brackets, S.E.M. Correlation coefficient (r) is highly significant. Definite myocardial injury is found when the average (integrated) catecholamine concentration exceeds 1000 pg/ml.
significantly affected these parameters. Integrated mean values (averaged over the 90 min infusion period) indicate that practotol reduced the increases in both arterial pressure and heart rate. The P x R was markedly lower (P < 0.001). Phentolamine completely ablated the pressure rise, but did not modify the changes in heart rate. When both alpha and beta blocking drugs were given, heart rate and blood pressure responses to tyramine were abolished. T o be noted is the finding that the P x R product following practolol (21.4 __+2.4 x 103) did not differ from that after giving both blocking agents
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FIGURE 4. Total catecholamine concentrations measured at specified intervals during infusion of various doses of tyramine (#g/min/kg). Catecholamine release into the circulation is a function of tyramine dosage. [ A - - A , Tyramine 500; ~---s Tyramine 300; I - - l , Tyramine 200; D---[] Tyramine 0 (saline).]
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FIGURE 6. Effects of various adrenergic blocking agents on extent of myocardial injury following tyramine in highest dosage (Ts00). Results of pretreatment with practolol, (+P4), phentolamine (Regitine), (+Rio) or both blockers ( + P4 + Rl0) are shown. Myofiber injury was prevented by alpha but not beta blockade. Vertical brackets, S.E.M. Each group, n = 5.
Cardiac Injury from Endogenous
383
Catecholamines
TABLE 2. Responses to tyramine before and after adrenergic blockade Control
5 min.
BP
HR
BP • H R
BP
HR
Ts00 SE
104 2.6
252 13
26.1 1.4
158 8.7
+P4 SE
93 4.0
234 10
21.8 1.9
+Rto SE
97 3.5
246 I!
24.0 1.5
+ P4 + Rt0 SE
91 4.6
250 18
22.8 2.7
Integrated mean BP • H R
BP
HR
BP • H R
239 35
37.8 6.2
126 6.4
283 20
38.4 3.1
137 9.1
150 37
21.7 6.8
108" 7.5
201" 21
21.4" 2.4
100 11
302 9.5
30.0 2.4
89" 8.6
299 12
26.8" 1.3
192 16
17.2* 1.9
86" 7.5
210" t2
17.9 a 0.5
90" 8.2
B ~ m e a n arterial pressure (mmHg). HR--heart rate beats/min. BP x HR--double product (10- 3). Tsoo--tyramine, 500 #g/min/kg (90 min). P4--practolol (4 mg/kg). Rlo--Regitine (phentolamine, 10 mg). Control values after giving blocking agents. a p < 0.05 v. Tso o . (17.9 + 0.5 x 103). T h u s m y o c a r d i a l injury was likely i n d e p e n d e n t of m e t a b o l i c d e m a n d . C a t e c h o l a m i n e concentrations were determ i n e d in three a d d i t i o n a l animals given tyramine (Ts00) after pretreatment with p h e n t o l a m i n e (Fig. 7). A t each s a m p l i n g i n t e r v a l d u r i n g t y r a m i n e infusion the total 3000 i ,..a
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FIGURE 7. Changes in total circulating catecholamine concentrations at various intervals during tyramine infusions (A--A) (500 ktg/min/kg) in animals without (closed triangles, n = 5) or with phentolamine blockade (m--D) (closed squares, n = 3). Concentrations were significantly lower in phentolamine treated animals, but remained in range sufficient to cause myofiber injury (see text). * P < 0.05; ** P < 0.001.
c a t e c h o l a m i n e concentrations were significantly lower than in those not given p h e n t o l a mine. T h e i n t e g r a t e d m e a n value was also less (1520 • 171 pg/ml, c o m p a r e d with 2445 • 270 pg/ml).
Effects of insulin excess and deficiency on myocardial injury following tyramine administration Five a n i m a l s were given insulin (10 U/kg) 30 min before c o m m e n c i n g t y r a m i n e infusion. Blood glucose values m e a s u r e d at initiation of infusion a v e r a g e d 133 m g / d l in controls a n d 104 mg/gl in those given insulin. As shown in Figure 8, the extent of m y o c a r d i a l injury in those given insulin was significantly lower ( P < 0.05) than those not pretreated. T h e m e a n histological score was reduced to 0.99 (+0.29). I n view o f the protective effect of exogenous insulin a d m i n i s t r a t i o n , studies were pursued to d e t e r m i n e if insulin deficiency enhances m y o c a r d i a l sensitivity to t y r a m i n e injury. D a t a from ten rabbits given alloxan are shown in the right p a n e l o f Figure 8. Blood glucose concentrations a v e r a g e d 277 mg/dl, c o m p a r e d with n o n - d i a b e t i c controls which a v e r a g e d 151 mg/dl (P < 0.001). T h e m e a n histological score from animals given the lowest dose of t y r a m i n e (200 #g/min/kg,
384
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277
F I G U R E 8. Effects of insulin and experimental diabetes on myocardial sensitivity to tyramine-induced
injury. Tyramine 500 (Tso0) or 200 (T2oo) #g/min/kg was given after 10 units ofinsulin/kg (Ilo), or to alloxan diabetic animals (T2oo + A), respectively. Insulin reduced myocardial injury from high doses of tyramine (a). However, insulin deficiency (diabetes) did not enhance the extent of myofiber damage following low doses of tyramine (b). Vertical brackets, s.E.~.
90 min) was 0.80 (4-0.25). A virtually identical score ( 0 . 8 2 _ 0.20) was obtained in the diabetic group. Thus no evidence f o r increased injury propensity from endogenously released catecholamines was found in the insulin deficient animals.
Discussion
It may be concluded from the present study that endogenous release of catecholamines engendered by tyramine causes patterns of myocardial injury identical with those following exogenous norepinephrine infusion [4, 5]. Moreover, the severity of lesion production as judged by the histological scoring method is a function of the dose of tyramine given (Fig. 3). Measurements of total circulating catecholamine concentrations over the time course studied indicate a positive correlation with the amount of tyramine given (Fig. 4). These relationships are verified by a highly significant correlation (r = 0.842) between the average catecholamine concentration and the histological score as depicted in Figure 5. This analysis also attests to the accuracy of the two very different systems for quantification of these variables. Total catecholamine concentrations in the control state averaged 450 pg/ml, and were unchanged during a 90 min saline infusion (Fig. 4). Myocardial lesions
were totally absent in the latter group. Most severe cardiac injury appeared when catecholamine levels average 2000 pg/ml (Fig. 5). Concentrations measured in two additional animals given NE, 2 #g/min/kg (90 rain), were much higher, averaging over 14,000 pg/ml. The histological score in each animal was 1.75. Thus it appears that maximal injury potential is achieved at substantially lower concentrations in this model. Mechanisms responsible for the cardiomyopathic changes induced by the catecholamines have been under intense study. The possibility that a supply-demand imbalance occasioned by sustained high level inotropic and chronotropic stimulation is responsible appears unlikely on several counts. In these studies the pressure-rate product (P x R), which has repeatedly been shown to closely reflect myocardial oxygen consumption [25, 26], increased substantially during tyramine infusion (Fig. 1, Table 2). However, in animals subjected to beta adrenoceptor blockade, the P x R product remained unchanged during tyramine administration. The histological scores in these two groups were identical, indicating no relationship to oxygen demand. This is consistent with our earlier work showing that the P x R product remains unchanged in animals given NE sufficient to cause near maximal histological damage (2 #g/min/kg), and does not differ from animals given saline [5]. Others have suggested that the lesions may be fundamentally ischemic in nature, perhaps due to vascular injury or the appearance of platelet thrombi [12]. Against this view is the fact that the cellular infiltrate is mononuclear and granulocytes are extremely rare. Two days after an ischemic event the infiltrate would be predominantly polymorphonuclear leukocytes. Secondly, platelet thrombi have never been encountered in our experience with several hundred animals examined in this and earlier [4, 5] studies. Myocardial hemorrhage, which would be expected with microvascular injury leading to an ischemic process, was also absent. It has generally been assumed that should adrenergic receptor activation be a fundamental mechanism for the production of catecholamine cardiomyopathy, the beta 1adrenoceptors would likely be involved.
Cardiac Injury from Endogenous Catecholamines
Indeed, focal myocardial lesions have been described in other species given massive doses of isoproterenol [24]. However, this does not appear to be the case, at least for the rabbit. Beta receptor blockade with practolol (4 mg/kg), or with propranolol is without effect in preventing cardiac injury by norepinephrine [5]. This is consistent with findings in the present study where practolol also failed to inhibit lesion formation following tyramine (Fig. 6). On the other hand, alpha adrenoceptor blockade with phentolamine effectively eliminated myocardial injury in both studies. The increase in circulating catecholamines engendered by tyramine was for reasons which remain obscure significantly less in animals pretreated with phentolamine (Fig. 7). Nevertheless, average concentrations of 1520 ( + 171) pg/ml would be sufficient to cause significant myocardial injury, as indicated by the data in Figure 5. Thus it is most likely that blockade of myocardial alpha receptor-agonist binding was primarily responsible for the prevention of myofiber injury in this, as well as our previous study using NE. The use of phentolamine does not distinguish the alpha receptor sub-type, however. The conclusion that activation of alpha adrenoceptor pathways is an obligatory factor is further supported by the finding that identical myocardial injury patterns result from administration of the pure alpha agonist, methoxamine [5]. This also occurs in a dosedependent manner. While these results might not have been predicted on first consideration, there is growing evidence for species specific variation in myocardial alpha receptor sensitivity. Earlier studies have shown that methoxamine elicits significant positive inotropic responses in cat (21), rabbit (7), rat (19) and lamb (16), but not in dog hearts (2). This inconsistency is likely explained by the recent observations of Mukherjee et al. [18]. These workers found a markedly reduced number of alpha 1 receptors and increased number of beta receptors in dog heart sarcolemma compared with rat and rabbit. Binding properties were similar in all species. Thus the widely used dog model differs substantially in this regard from several other species in which myocardial alpha 1 receptor populations are probably substantially greater.
385
We have previously shown that pretreatment of rabbits with large doses of insulin (10 U/kg) substantially prevents the cardiomyopathic changes produced by NE [4, 32]. Insulin also sharply reduces contractility responses to NE in isolated cardiac muscle [17] and intact heart preparations [20]. Findings in the present study (Fig. 8, left panel) are consistent with these prior observations. Pretreatment with insulin significantly reduced the severity of myocardial damage from tyramine administration. The reduction was quantitatively identical with that seen in studies using NE [4, 32], further supporting the assumption that tyramine acts exclusively via catecholamine release. In view of the protective effects of exogenous insulin, we raised the possibility that insulin-deficient diabetics might be more prone to catecholamine cardiomyopathy [4]. Moreover, we have recently shown that inotropic sensitivity of diabetic hearts to alpha adrenoceptor stimulation is enhanced [6]. The present study failed to support this hypothesis, however. Using the lower dose of tyramine (200 ffg/min/kg), the average histological scores in controls and diabetic rabbits were identical (Fig. 8). This is consistent with our inability to demonstrate a greater propensity for NE-induced injury in diabetic rabbits (unpublished observations). It is also in agreement with the observations of Fein et al. [9] who used a streptozotocin diabetic rat model. Myocardial damage resulting from either isoproterenol or NE was identical with controls in this model as well. From these three lines of evidence it appears unlikely that insulin deficiency sufficient to cause moderate or severe [9] hyperglycemia increases myocyte vulnerability to catecholamine injury. It may be concluded from the present study that endogenous release of catecholamines is capable of inducing significant cardiomyopathic changes in the rabbit. Close positive correlations between the amount of tyramine given, resulting plasma catecholamine concentrations and the extent of lesion generation judged by histological analysis have been demonstrated. As was found with exogenously administered agonists, activation of alpha adrenergic pathways is the dominant mechanism in the rabbit model. Still to be resolved is whether myocyte injury is solely the conse-
386
S. Evans D o w n i n g and V. Chen
q u e n c e o f m e m b r a n e r e c e p t o r a c t i v a t i o n , or w h e t h e r i n d i r e c t effects of i n c r e a s e d a f t e r l o a d or c o r o n a r y v a s o c o n s t r i c t i o n [28] are c o n t r i b utory. A g o n i s t a c t i v a t i o n of a l p h a 1 receptors enhances phosphotidylinositol turnover with release of b o u n d i n t r a c e l l u l a r C a 2 +, as well as i n c r e a s e d u p t a k e o f e x t r a c e l l u l a r C a 2§ [8]. It is t e m p t i n g to s p e c u l a t e t h a t in excess this m a y trigger a c c e l e r a t e d d e g r a d a t i o n of m e m b r a n e p h o s p h o l i p i d s as d e s c r i b e d in i s c h e m i a [3-]. T h e l a t t e r is followed by m a r k e d increases in m y o c a r d i a l C a 2§ c o n c e n t r a t i o n a n d a m a n y - f o l d increase in passive C a 2+ perm e a b i l i t y of s a r c o p l a s m i c r e t i c u l u m . R e gardless, it is well established t h a t C a 2§
o v e r l o a d i n g is a f e a t u r e of c a t e c h o l a m i n e i n d u c e d m y o f i b e r i n j u r y [3, 10]. Precise deline a t i o n of m e c h a n i s m s a n d sequences m u s t await further investigation.
Acknowledgements T h e a u t h o r s are g r a t e f u l for the e x c e l l e n t t e c h n i c a l assistance p r o v i d e d by R o n a l d Gordon, William Stronk and Sandra Rancourt. D r C o l i n W h i t e p r o v i d e d v a l u a b l e h e l p w i t h statistical t r e a t m e n t of the data. S u p p o r t e d in p a r t by g r a n t s H L - 2 0 4 0 1 a n d H L - 0 8 6 5 9 f r o m the N a t i o n a l Institutes of Health.
References 1 BLOOM,S., DAVIS,D. L. Calcium as mediator of isoproterenol-induced myocardial necrosis. Am J Pathol 69, 459 470 (1972). 2 BREWSTER,W. R., JR, OS~OOD, P. F., ISAACS,J. P., GOLDBERG,L. I. Hemodynamic effects of a pressor amine (methoxamine) with predominant vasoconstrictor activity. Circ Res 8, 980 988 (1960). 3 CmEN, K. R., PVAU,R. G., FARBER,J. L. Ischemic myocardial cell injury. Prevention by chlorpromazine of an accelerated phospholipid degradation and associated membrane dysfunction. Am] Patho197, 505 530 (1979). 4 DOWNING,S. E., LEE, J. C. Effects of insulin on experimental catecholamine cardiomyopathy. Am J Pathol 93, 339-352 (1978). 5 DOWNING,S. E., LEE, J. C. Contribution of ~t-adrenoceptor activation to the pathogenesis of norepinephrine cardiomyopathy. Circ Res 52, 471-478 (1983). 6 DOWNING,S. E., LEE, J. C., FRIPP, R. R. Enhanced sensitivity of diabetic hearts to a-adrenoceptor stimulation. AmJ Physio1245 (Heart Circ Physiology 14), H808 H813 (1983). 7 ENDOH,M., SCHUMANN,H.J. Frequency-dependence of the positive inotropic effect of methoxamine and naphazoline mediated by alpha-adrenoceptors in the isolated rabbit papillary muscle. Nauyn-Schmiedebergs Arch Pharmaco1287,377-389 (1975). 8 FAIN, J. C., GARCIA-SAINZ,J. A. Role of phosphatydylinositol turnover in alpha 1 and of adenylate cyclase inhibition of alpha 2effects of catecholamines. Life Sci 26, 1183-1194 (1980). 9 FE1N, F. S., FACTOR, S. M., CHO, S., MILLER-GREEN, B., WORTSMANNCARROLL, D., SONNENBLICK,E. H. Catecholamine-induced myocardial necrosis in experimental diabetes mellitus. Arch Pathol Lab Med 107, 480-483 (1983). 10 FLECKENSTEIN,A., JAN,:E, J., D/SRINa, H. J., LEDER, O. Myocardial fiber necrosis due to intracellular Ca overload a new principle in cardiac pathophysiology. In : Recent Advances in Studies on Cardiac Structure and Metabolism. Myocardial Biology, N. S. Dhalla (Ed.), vol. 4, pp. 563-580. Baltimore: University Park Press (1974). 11 FERRANS,V.J., HIBBS,R. G., WALSH,J. J., BURCH,G. E. Histochemical and electron microscopical studies on the cardiac necroses produced by sympathomimetic agents. Ann NY Acad Sci 156, 309-332 (1969). 12 HAFT,J. I. Cardiovascular injury induced by sympathetic catecholamines. Progress in Cardiovascular Diseases 17(1), (1974). 13 HOAK,J. C., WARNER, E. D., CONNER,W. E. New concept of levarterenol-induced acute myocardial necrosis. Arch Patho187, 332-338 (1969). 14 KLINE,I. K. Myocardial alterations associated with pheochromocytomas. AmJ Patho138, 539 557 (1961). 15 KRAIKITPANITCH,S., HAYGOOD,C. C., BAXTER,D. J., YUNICE,A. A., LINDEMAN,R. D. Effects of acetylsalicylic acid, dipyridamole,and hydrocortisone on epinephrine-induced myocardial injury in dogs. Am Heart J 92, 615 622 (1976). 16 LEg, J. C., FRIPP, R. R., DOWNING,S. E. Myocardial responses to a-adrenoceptor stimulation with methoxamine hydrochloride in lambs. AmJ Physio1242 (Heart Circ Physio111), H405-H410 (1982). 17 LEE,J. C., DOWNING,S. E. Effects of insulin on cardiac muscle contraction and responsiveness to norepinephrine. AmJ Physio1230, 1360 1365 (1976). 18 MUKHERJEE,A., HAGHANI,Z., BRADY,J , BUSH,L., McBRIDE,W., BUJA,L. M., WILLERSON,J. T. Differences in myocardial ~- and ~-adrenergic receptor numbers in different species. Am J Physiol 245 (Heart Circ Physiol 14), H957-H961 (1983). 19 NAKASHIMA,M., TSURU,H., SHIEGEI,T. Stimulant action of methoxamine in the isolated atria of normal and 6-propyl-2-thiouracil-fed rats. J p n J Pharmacol Ther 23, 307 312 ( 1973).
Cardiac Injury from Endogenous Catecholamines 20 21 22 23 24 25 26 27 28 29 30 31 32 33
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NUDEL, D. B., LE~, J. C., DOWNING, S. E. Reciprocal inhibition of cardiac responses to norepinephrine and insulin. AmJ Physio1233, H665 H669 (1978). RABINOWn'Z,B., CHUCK, L., KLIOERMAN, M., PARMLEY, W. W. Positive inotropic effects of methoxamine: evidence for alpha-adrenergic receptors in ventricular myocardium. A m J Physio1229, 582--585 (1975). REEVES,W. C., JACKSON, G. L., FLICKINOER, F. W., KWEE, H. G., SCHWITeR, E.J., WERNER, J., WHITSELL, L., BIDDLE, M. A., COPENHAVER, G., SHAIKH, B. S., ZELIS, R. Radionuclide imaging of experimental myocarditis. Circulation 63, 640-644 (1981). REICHENBACH, D. D., BEND1TT, E. P. Catecholamines and cardiomyopathy: the pathogenesis and potential importance ofmyofibrillary degeneration. Hum Pathol l, 125 150(1970). RONA,G., KAHNA, D. S., C:4APPEL, C. 1. Studies on infarct-like myocardial necrosis produced by isoproterenol: a review. Rev Can Bio122, 241-255 (1963). ROOKE,G. A., FEraL, E. O. Work as a correlate of canine left ventricular oxygen consumption, and the problem of catecholamine oxygen wasting. Circ Res 50, 273-286 (1982). SARNOFF,S. J., GILMORE,J. P., WEISFELDT,M. L., DAGGETT,W. M., MANSFIELD,P. B. Influence of norepinephrine on myocardial oxygen consumption under cotrolled hemodynamic conditions. Am J Cardiol 16, 217 226 (1965). SCHENK,E. A., Moss, A.J. Cardiovascular effect of sustained norepinephrine infusion. If. Morphology. Circ Res 18, 605-615 (1966). StMONS,M., DOWNING, S. E. Coronary vasoconstriction and catecholamine cardiornyopathy. Fed Proc 43, 423 (1984). SZAKACS,J. E., CANYON,A. l-Norepinephrine-myocarditis. Am J Clin Patho130, 425 434 (1958). VAN VLIET, P. D., BC'RCHELC,H. B., Trrus, J. L. Focal myocarditis associated with pheochromocytoma. N EnglJ Med 272, 1102-1108 (1966). WATKINS,D. B. Pheochromocytoma: A review of the literature.J Chronic Dis 6, 510 527 (1957). WERNER,J. C., LEE, J. C., DOWN1Na, S. E. Preservation of left ventricular function by insulin and experimental catecholamine cardiomyopathy. A m J Physio1238, H257 H262 (1980). ZAR,J. H. Biostatistical Analysis. W. D. McElroy, C. P. Swanson (Eds.), Englewood Cliffs, NJ: Prentice-Hall, Inc (1973).
Myocardial Injury Following Endogenous Catecholamine R e l e a s e in Rabbits S. Evans Downing and Victor Chen Department of Pathology, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06510, USA (Received I June 1984, accepted in revisedform 7 September 1984) S. EvAns Dow~I~G AND V. CIaEN. Myocardial Injury Following Endogenous Catecholamine Release in Rabbits. Journal of Molecular and Cellular Cardiology (1985) 17, 377 387. Catecholamines (CAT) given in large doses produce cardiomyopathic changes in several animal species. This study was designed to determine if endogenous release can also induce cardiac injury, Rabbits were infused with doses of tyramine (TYR), ranging from 200 to 500 #g/min/kg, i.v. for 90 rain. Arterial pressure and heart rate were measured, as were total C A T concentrations, blood gases, pH and glucose. Two days later the animals were killed and cardiac injury assessed using a histological scoring system. All data were compared with controls given saline. Initial C A T averaged 452 pg/ml, rose to 2890 pg/ml after starting TYR, 500 ktg/min/kg, and remained elevated for the duration of infusion. Circulating C A T levels were a function of T Y R dose, and bore a linear relationship to the histological score (P < 0.001). Development of lesions was unaltered by betaj blockade with practolol, but sharply reduced by alpha blockade with phentolamine (P < 0.01). Pretreatment with insulin also reduced lesion formation, but diabetic (alloxan) rabbits showed no greater C A T injury. It is concluded that endogenous release of C A T induces myocardial injury in the rabbit in a dose-dependent manner. This is unrelated to myocardial O 2 demand, and microvascular pathology was absent. Activation of alpha adrenergic pathways is likely the dominant or exclusive mechanisml KEY WORDS: Cardiomyopathy; Tyramine; Catecholamines; Rabbits; Practolol; Phentolanfine; Alloxan diabetes ; Insulin ; Cardiac O2demand ; Cardiac muscle necrosis.
Introduction wtlich provides a consistent and reproducible Exogenous administration of large doses of cardiomyopathy with limited quantitative catecholamines is known to cause myocardial variability as judged by a histological scoring damage in several animal species [4, 11, 13, 15, system [4]. Using a constant time base of 90 23, 24, 27, 29]. Similar patterns of cardiac rain, infusions of relatively modest doses of NE injury have been described in humans as well (2 to 3 /2g/min/kg) is sufficient to produce [10, 23, 29]. Several adrenergic agonists have extensive myofiber injury judged by light been employed in experimental studies, and microscopy and also by radionuclide cardiac these have yielded similar results. Thus nor- imaging [22]. Moreover, the magnitude of epinephrine [4, 5, 11, 13, 23], epinephrine [11, cardiac injury is dose-related [5]. Physiologi23] and isoproterenol [1, 9, 24] have all been cal studies have demonstrated substantial found to cause myofiber injury with appar- impairment of left ventricular performance in ently identical histological patterns. While this model [17, 32]. this serves as a common denominator, interWhereas previous work establishes that the pretation of the findings and possible patho- catecholamines given in large doses leads to genetic mechanisms have been confounded by important cardiomyopathic changes, it is less widely varying protocols, and doses which are clear whether endogenous catecholaminc release is potentially injurious. The exception often massive [-1]. Based on earlier studies by Schenk and is data from human subjects with malignant Moss [27], we have developed a rabbit model pheochromocytoma [14, 29, 30, 31]. A limited 0022-2828/85/040377 + 11 $03.00/0
9 1985 Academic Press Inc. (London) Limited
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S. Evans D o w n i n g and V. Chen
number of these patients exhibited lesions similar to those found in experimental catecholamine injury. However, these are rare cases and do not address the question of potential myofiber injury by circulating catecholamines released endogenously in the normal organism. The objectives of the present study were to determine in the rabbit model if endogenous release of catecholamines using tyramine can elicit myocardial damage ; to establish dose-response relations; to measure changes in circulating catecholamines and their relationship to lesion formation; and to determine the receptors primarily responsible by using adrenoceptor blocking agents. The question of enhanced sensitivity to myofiber damage in the insulin deficient state [4] was also explored in animals with alloxan diabetes.
phentolamine (Ciba-Geigy), 2.5 mg/kg. Insulin (10 U/kg) was given to five animals 30 min before tyramine infusion was begun. Effects of insulin deficient diabetes mellitus on tyramine-induced myocardial injury was studied in ten animals given altoxan monohydrate (Sigma), 100 to 110 mg/kg, as an i.v. bolus injection. Tyramine infusions were performed 10 to 40 days after alloxan treatment. Arterial blood samples were obtained before, and after 5, 15, 60 and 90 min oftyramine infusion to determine circulating catecholamine concentrations. Total plasma catecholamines were measured with a commercially available kit (Cat-A-Kit, Upjohn Diagnostics). The radioenzymatic assay involves the transfer of a [3H]methyl group from S-adenosyl-L-methionine-[3H]methyl to epinephrine or norepinephrine which is catalyzed by the enzyme catechol-o-methyltransferase. The resulting [3H]methoxy Methods derivatives are oxidized to [3H]vanillin, Data to be presented in this study were extracted and quantified by liquid scintilobtained from 74 New Zealand white rabbits lation counting. of either sex, and with a mean body weight of Following infusion the catheters were 4.0 kg. All animals were anesthetized with removed, the femoral wound surgically closed pentobarbital, 30 mg/kg and polyethylene and the animals returned to their cages after catheters were placed in a femoral artery and recovery from anesthesia. They were fed a vein. Arterial pressure was continuously mea- standard diet and water ad libitum. All animals sured with a Sanborn transducer and heart were killed 2 days later by an overdose of rate determined with a Sanborn cardio- pentobarbital via an ear vein. The hearts were tachometer. The latter was verified by immediately removed, emptied and weighed. manual assessment of pulse frequency from The atria and R V free wall were dissected and pressure traces inscribed by a Sanborn weighed, and the LV ( + s e p t u m ) separately (Model 7700) recorder. Body temperature weighed. Transverse 'ring' sections of LV was maintained with a heating pad. Arterial were obtained from the basal and mid porblood samples were obtained at 15 min inter- tions and fixed in 10% buffered formalin. vals and analyzed for Po2, Pco2 and p H They were prepared by standard histological using an Instrumentation Laboratories methods and stained with hematoxylin and analyzer. Hematocrit and glucose concentra- eosin for subsequent analysis. tions (Glucostat, Worthington Biochemical) Morphological evaluation employed a were also determined. semi-quantitative histological scoring system Tyramine (Sigma) was freshly prepared by described previously [4]. In brief, each section dilution in normal saline to concentrations was graded by two observers according to the that would provide the desired dose when extent and intensity of the leukocytic infused at a constant rate of 0.382 ml/min. response, without prior knowledge of the proDoses of 500, 400, 300 or 200 #g/min/kg (free cedures used in a given animal. A maximum base) were given i.v. for 90 min. Control score of 2.0 was given when the lesions were animals were similarly infused with 0.9% florid, extensive and transmural. Those with saline. In selected experiments adrenoceptor definite but sparse lesions were scored 1.0. blocking agents were given i.v. 10 rain prior to Equivocal focal lesions were scored 0.5. Those initiation of tyramine infusion. These judged to manifest injury more extensive than included practolol (Ayerst), 4 mg/kg; and 1.0, but less than 2.0 (e.g. nontransmural)
Cardiac Injury f r o m E n d o g e n o u s C a t e c h o l a m i n e s
were assigned a score of 1.5. A score of 0 was given when no histologic abnormality was present. Values for each of the two sections were averaged and used in scoring a given heart. Substantial alterations in arterial pressure and heart rate occurred during tyramine administration. Maximal changes and integrated mean values were determined from data obtained at 10 rain intervals. The pressure-rate product was calculated as an index of metabolic demand [25, 26]. All data were analyzed by standard statistical methods using the non-paired Student t-test and presented as mean values • of the mean. For multiple comparisons, one-way analysis of variance followed by the Scheffb test was employed [33]. Differences were considered significant when P was < 0.05.
Results
Circulatory responses to tyramine Average changes in arterial pressure, heart rate and the pressure-rate product measured in animals given tyramine, 500 #g/min/kg, are ~
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379
shown in Figure 1. Mean arterial pressure (MAP) rose from 104 (__+2.6) m m H g to 148 (___9.5) m m H g within 2 rain. After 10 min it began to decline, and at 30 min had fallen to 115 (-t-7) m m H g , not significantly greater than initial control values. At 90 min, MAP was 86 (+2.1) m m H g , significantly lower than control values. In contrast, heart rate increased from 252 ( + 13) beats/rain to 330 ( + 1 1 ) at 10 min, and remained elevated throughout the duration of tyramine infusion. Saline infused animals showed no significant hemodynamic changes (Fig. 1, open circles). The pressure-rate (P x R) product (lower panel) increased substantially with tyramine infusion from 26.1 (-t-1.4) to 48.7 (-t-1.9) units and remained elevated throughout most of the infusion period. The mean P x R for the tyramine group was 34.8 (_+ 3.1), compared with the saline group which averaged 25.2 (_+ 0.6) (P < 0.05). Thus, maximal and average myocardial 0 2 requirements were likely substantially greater in the animals given tyramine. Metabolic measurements obtained for each group are listed in Table 1.
Morphological patterns of i n jury T h e characteristic pattern of myocardial injury resulting from infusion of larger doses of tyramine is illustrated in Figure 2. A heavy cellular infiltrate of predominantly mononuclear cells was uniformly present. Large histiocytic cells were most numerous, accompanied by less frequent lymphocytes. Granulocytes, including eosinophils, were occasionally present. The infiltrate was interstitial, and tended to concentrate in association with foci of myofiber necrosis. In addition to fragmentation and focal myofiber destruction, numerous contraction bands and zones of granularity consistent with swollen mitochondria were also evident with light microscopy. Z-lines were generally indistinct and myofiber nuclei were often lost in the more active inflammatory foci. These changes were in general most pronounced in the papillary muscles and inner half of the ventricular wall. However, a transmural distribution was often observed in hearts subjected to higher doses of tyramine. There was no distinction in the intensity of free wall or septal involvement. Neither the larger coronary arteries nor myocardial arterioles exhibited discernible
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pH
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Hematocrit (%~
Glucose (mg/dl)
Time (min)
Saline
Tyramine-200
Tyramine-300
Tyramine-500
0 30 60 90 100 0 30 60 90 100 0 30 60 90 I00 0 30 60 90 100 0 30 60 90 100
7.37 + 0.02 7.44 +_ 0.03 7.47 __ 0.03 7.48 ___0.03 7.48 • 0.02 62.7+ 5.7 73.0 __+5.6 78.3 • 5.9 79.3 • 9.6 79.0 • 6.0 39.3 _ 2.9 33.7 • 2.5 30.7 _+ 1.8 29.0 • 1.9 29.7 • 3.2 38.0 • 1.9 36.3 • 2.9 36.7 + 2.0 35.3 ___2.3 35.0 • 1.9 147 +_ 11 157 _ II 157 • 11 160 • t4 153 • 15
7.42 _+ 0.02 7.48 + 0.04 7.50 + 0.03 7.51 • (1.03 7.50 • 0.02 68.4 • 4.3 72.4 • 4.2 80.2 + 3.1 83.8 • 1.6 88.6 ___ 1.8 40.2 • 1.3 32.6 • 1.4 30.2 • 1.1 28.4 + 2.0 24.8 • 0.8 43.6 • 1.0 41.6 • t.3 42.8 • 1.2 41.6 • 0.6 38.4 ___0.6 158 -t- 14 168 • 17 170 + 22 176 • 22 188 • 25
7.41 _+ 0.02 7.52 + 0.03 7.56 +__0.02 7.55 __+0.01 7.51 +_ 0.03 56.2 __+3.4 64.4 • 3.0 73.2 __ 4.7 82.8 __ 4.9 86.8 • 5.1 43.6 • 3.5 34.6 -t- 2.9 31.6 __+2.6 28.0 • 2.5 24.6 +_ 2.8 40.8 __+0.8 41.2 • 1.0 42.4 • 1.0 41,5 • 1.7 39,0 • 1.3 166 • 19 200 + 20 194 • 17 174• 21 224 _ 18
7.43 +_ 0.01 7.48 • 0.08 7.53 _+ 0.08 7.51 +__0.07 7.51 + 0.07 63.2 ___5.6 51.7 + 3.5 57.8 __ 3.5 50.5 __ 12.3 58.2 • 14.4 40.0 _ 1.5 37.3 _ 1.1 35.7 • 2.1 31.2 __+3.3 27.5 + 3.6 41.5 _ 0.9 40.3 • 1.9 42.0 _ 4.4 41.2 • 4.3 38.0 • 5.1 140 --4-9 225 • 16 235 • 20 202 +_ 28 230 + 33
pg/ml (n = 17). Five minutes after b e g i n n i n g tyramine, 200/~g/min/kg, the average concentration rose to 1573 (___373) pg/ml. This m a y be contrasted with t y r a m i n e given at the rate of 500 #g/min/kg. I n this group the increase after 5 m i n was nearly twice as great {2889 _ 209 pg/ml). After 15 min total catecholamine concentrations declined somewhat (Fig. 4), b u t in all groups values remained significantly higher t h a n saline controls for the entire 90 m i n infusion period. A highly significant relationship was found between the histological score applied to each heart a n d average catecholamine concentrations. These data are shown in Figure 5. Linear regression analysis demonstrated a correlation coefficient of 0.842 ~P < 0.001 ~. Circulating catecholamine concentrations Integrated mean values (averaged over 90 during tyramine administration rain) for total catecholamine concentrations Effects of infusing various concentrations of in excess of 1000 pg/ml resulted in significant t y r a m i n e on total circulating catecholamine myofiber damage. Concentrations of approxlevels are shown in Figure 4. Initial m e a n imately 2000 pg/ml were associated with values in each group were less t h a n 500 pg/ml, m a x i m a l myocardial injury as j u d g e d by the a n d concentrations averaged 451.5 (__+_26] histological scoring system.
histopathological changes. T h r o m b i were never e n c o u n t e r e d in 150 sections examined. T h e m e a n histological scores o b t a i n e d from 38 rabbits infused with various a m o u n t s of t y r a m i n e are shown i n Figure 3, and illustrate the dose response relationship. Scores ranged from 1.84 (__+0.06) in a n i m a l s given 500 # g / m i n / k g to 0.97 (-t-0.17) in those gxven 200 # g / k g / m i n ~P < 0.001 ). Eight animals infused with saline showed no definite lesions ( m e a n score, 0.16 ___0.10). Regression analysis revealed a correlation coefficient of 0.848 ( P < 0 . 0 0 1 1 . Analysis of variance d e m o n strated that the group means differ (P < 0.05).
Cardiac Injury from Endogenous Catecholamines
381
/
FIGURE 2. (a). Photomicrograph of a typical histological section of left ventricular myocardium from a rabbit sacrificed 2 days after giving tyramine, 400 pg/min/kg for 90 min. H + E stain. Original magnification x 120. Intense interstitial cellular infiltrate, and myofiber separation and lossis evident. (b). Higher magnification (x 300). Myofiber vacuolization, granularity and degeneration is marked. The infiltrate is mononuclear, and consists mainly of cells with morphological features of histiocytes. Granulocytes are absent. This section was assigned a histological score of 2.0.
Effects of specific adrenoceptor blocking agents T o confirm that myofiber injury following t y r a m i n e was r e l a t a b l e to elevations in circulating catecholamines a n d receptor binding, selective blocking agents were employed. Results from 20 animals are presented in Figure 6 a n d T a b l e 2. T h e m e a n histological score from five rabbits given t y r a m i n e only (Ts00) was 1.80 (__+0.09). A n a d d i t i o n a l five
animals were p r e t r e a t e d with large doses (4 mg/kg) o f the beta I blocking agent, practolol ( + P 4 ) , p r i o r to initiation o f the t y r a m i n e infusion. T h e m e a n histological score of this g r o u p was 1.50 ( + 0.18), i n d i c a t i n g a persistent high level o f m y o c a r d i a l injury that d i d not differ from the group w i t h o u t beta blockade. This m a y be contrasted with animals p r e t r e a t e d with p h e n t o l a m i n e ( q - R i 0 ) in which the m e a n score was reduced to 0.53
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I I00
0
I __ I 200 300
400
I 50(
(Saline) Tyromine -p.cj/min / kg (90rnin) FIGURE 3. Relationship between tyramine dose and quantitative estimate of histological damage. Correlation coefficient, (r=0.848), is highly significant, slope, m = 0.344( 10- 2), intercept, b = 0.144. Vertical brackets, S.E.M. P < 0.001.
(-t-0.28). This difference was significant (P < 0.01) when compared with either of the preceding groups and indicates minimal or questionable injury. Similarly, in the five animals pretreated with both practolol and phentolamine ( + P 4 + Rt0) no significant lesions were identified, and the mean score was 0.05 ( + 0.02). This group differed significantly from the first two (P < 0.001), but not from the third group given phentolamine alone (-t-R10 ). These data indicate that myocardial injury following tyramine is largely or entirely relatable to activation of alpha adrenoceptor pathways, with little or no contribution from beta receptor activation. Circulatory changes summarized in Table 2 demonstrate that both blocking agents
0
0
015 I,O
115 2~-0
Histologicol score
FIGURE 5. Relationship of average catecholamine concentrations during tyramine infusion period (90 rain) to histological scores of hearts obtained at sacrifice 2 days later. Data include 70 catecholamine measurements in 18 rabbits. Vertical brackets, S.E.M. Correlation coefficient (r) is highly significant. Definite myocardial injury is found when the average (integrated) catecholamine concentration exceeds 1000 pg/ml.
significantly affected these parameters. Integrated mean values (averaged over the 90 min infusion period) indicate that practotol reduced the increases in both arterial pressure and heart rate. The P x R was markedly lower (P < 0.001). Phentolamine completely ablated the pressure rise, but did not modify the changes in heart rate. When both alpha and beta blocking drugs were given, heart rate and blood pressure responses to tyramine were abolished. T o be noted is the finding that the P x R product following practolol (21.4 __+2.4 x 103) did not differ from that after giving both blocking agents
3000 2"0
$ g
2000
1.5
r
E o
1.0
I
IO00 o
0.5 P'~O.OOI i
0 5
i
J
15
30
I
J
60
90
Time (rain)
FIGURE 4. Total catecholamine concentrations measured at specified intervals during infusion of various doses of tyramine (#g/min/kg). Catecholamine release into the circulation is a function of tyramine dosage. [ A - - A , Tyramine 500; ~---s Tyramine 300; I - - l , Tyramine 200; D---[] Tyramine 0 (saline).]
TSO0
*P4
+RIo
§
*RIo
FIGURE 6. Effects of various adrenergic blocking agents on extent of myocardial injury following tyramine in highest dosage (Ts00). Results of pretreatment with practolol, (+P4), phentolamine (Regitine), (+Rio) or both blockers ( + P4 + Rl0) are shown. Myofiber injury was prevented by alpha but not beta blockade. Vertical brackets, S.E.M. Each group, n = 5.
Cardiac Injury from Endogenous
383
Catecholamines
TABLE 2. Responses to tyramine before and after adrenergic blockade Control
5 min.
BP
HR
BP • H R
BP
HR
Ts00 SE
104 2.6
252 13
26.1 1.4
158 8.7
+P4 SE
93 4.0
234 10
21.8 1.9
+Rto SE
97 3.5
246 I!
24.0 1.5
+ P4 + Rt0 SE
91 4.6
250 18
22.8 2.7
Integrated mean BP • H R
BP
HR
BP • H R
239 35
37.8 6.2
126 6.4
283 20
38.4 3.1
137 9.1
150 37
21.7 6.8
108" 7.5
201" 21
21.4" 2.4
100 11
302 9.5
30.0 2.4
89" 8.6
299 12
26.8" 1.3
192 16
17.2* 1.9
86" 7.5
210" t2
17.9 a 0.5
90" 8.2
B ~ m e a n arterial pressure (mmHg). HR--heart rate beats/min. BP x HR--double product (10- 3). Tsoo--tyramine, 500 #g/min/kg (90 min). P4--practolol (4 mg/kg). Rlo--Regitine (phentolamine, 10 mg). Control values after giving blocking agents. a p < 0.05 v. Tso o . (17.9 + 0.5 x 103). T h u s m y o c a r d i a l injury was likely i n d e p e n d e n t of m e t a b o l i c d e m a n d . C a t e c h o l a m i n e concentrations were determ i n e d in three a d d i t i o n a l animals given tyramine (Ts00) after pretreatment with p h e n t o l a m i n e (Fig. 7). A t each s a m p l i n g i n t e r v a l d u r i n g t y r a m i n e infusion the total 3000 i ,..a
2000 o o
I00O o F--
I
I
0 5
I
I
]
I
15
30
60
90
Time (min)
FIGURE 7. Changes in total circulating catecholamine concentrations at various intervals during tyramine infusions (A--A) (500 ktg/min/kg) in animals without (closed triangles, n = 5) or with phentolamine blockade (m--D) (closed squares, n = 3). Concentrations were significantly lower in phentolamine treated animals, but remained in range sufficient to cause myofiber injury (see text). * P < 0.05; ** P < 0.001.
c a t e c h o l a m i n e concentrations were significantly lower than in those not given p h e n t o l a mine. T h e i n t e g r a t e d m e a n value was also less (1520 • 171 pg/ml, c o m p a r e d with 2445 • 270 pg/ml).
Effects of insulin excess and deficiency on myocardial injury following tyramine administration Five a n i m a l s were given insulin (10 U/kg) 30 min before c o m m e n c i n g t y r a m i n e infusion. Blood glucose values m e a s u r e d at initiation of infusion a v e r a g e d 133 m g / d l in controls a n d 104 mg/gl in those given insulin. As shown in Figure 8, the extent of m y o c a r d i a l injury in those given insulin was significantly lower ( P < 0.05) than those not pretreated. T h e m e a n histological score was reduced to 0.99 (+0.29). I n view o f the protective effect of exogenous insulin a d m i n i s t r a t i o n , studies were pursued to d e t e r m i n e if insulin deficiency enhances m y o c a r d i a l sensitivity to t y r a m i n e injury. D a t a from ten rabbits given alloxan are shown in the right p a n e l o f Figure 8. Blood glucose concentrations a v e r a g e d 277 mg/dl, c o m p a r e d with n o n - d i a b e t i c controls which a v e r a g e d 151 mg/dl (P < 0.001). T h e m e a n histological score from animals given the lowest dose of t y r a m i n e (200 #g/min/kg,
384
S. Evans D o w n i n g and V. Chen 2.0 3.1_
gt
t.5
(a)
(b)
P"=O" 0 5 " P=NS
I.O
0.5 I
TBoo Glucose: 1~3
Tsoo+Ito 104
T~OO Taoo+A 151
277
F I G U R E 8. Effects of insulin and experimental diabetes on myocardial sensitivity to tyramine-induced
injury. Tyramine 500 (Tso0) or 200 (T2oo) #g/min/kg was given after 10 units ofinsulin/kg (Ilo), or to alloxan diabetic animals (T2oo + A), respectively. Insulin reduced myocardial injury from high doses of tyramine (a). However, insulin deficiency (diabetes) did not enhance the extent of myofiber damage following low doses of tyramine (b). Vertical brackets, s.E.~.
90 min) was 0.80 (4-0.25). A virtually identical score ( 0 . 8 2 _ 0.20) was obtained in the diabetic group. Thus no evidence f o r increased injury propensity from endogenously released catecholamines was found in the insulin deficient animals.
Discussion
It may be concluded from the present study that endogenous release of catecholamines engendered by tyramine causes patterns of myocardial injury identical with those following exogenous norepinephrine infusion [4, 5]. Moreover, the severity of lesion production as judged by the histological scoring method is a function of the dose of tyramine given (Fig. 3). Measurements of total circulating catecholamine concentrations over the time course studied indicate a positive correlation with the amount of tyramine given (Fig. 4). These relationships are verified by a highly significant correlation (r = 0.842) between the average catecholamine concentration and the histological score as depicted in Figure 5. This analysis also attests to the accuracy of the two very different systems for quantification of these variables. Total catecholamine concentrations in the control state averaged 450 pg/ml, and were unchanged during a 90 min saline infusion (Fig. 4). Myocardial lesions
were totally absent in the latter group. Most severe cardiac injury appeared when catecholamine levels average 2000 pg/ml (Fig. 5). Concentrations measured in two additional animals given NE, 2 #g/min/kg (90 rain), were much higher, averaging over 14,000 pg/ml. The histological score in each animal was 1.75. Thus it appears that maximal injury potential is achieved at substantially lower concentrations in this model. Mechanisms responsible for the cardiomyopathic changes induced by the catecholamines have been under intense study. The possibility that a supply-demand imbalance occasioned by sustained high level inotropic and chronotropic stimulation is responsible appears unlikely on several counts. In these studies the pressure-rate product (P x R), which has repeatedly been shown to closely reflect myocardial oxygen consumption [25, 26], increased substantially during tyramine infusion (Fig. 1, Table 2). However, in animals subjected to beta adrenoceptor blockade, the P x R product remained unchanged during tyramine administration. The histological scores in these two groups were identical, indicating no relationship to oxygen demand. This is consistent with our earlier work showing that the P x R product remains unchanged in animals given NE sufficient to cause near maximal histological damage (2 #g/min/kg), and does not differ from animals given saline [5]. Others have suggested that the lesions may be fundamentally ischemic in nature, perhaps due to vascular injury or the appearance of platelet thrombi [12]. Against this view is the fact that the cellular infiltrate is mononuclear and granulocytes are extremely rare. Two days after an ischemic event the infiltrate would be predominantly polymorphonuclear leukocytes. Secondly, platelet thrombi have never been encountered in our experience with several hundred animals examined in this and earlier [4, 5] studies. Myocardial hemorrhage, which would be expected with microvascular injury leading to an ischemic process, was also absent. It has generally been assumed that should adrenergic receptor activation be a fundamental mechanism for the production of catecholamine cardiomyopathy, the beta 1adrenoceptors would likely be involved.
Cardiac Injury from Endogenous Catecholamines
Indeed, focal myocardial lesions have been described in other species given massive doses of isoproterenol [24]. However, this does not appear to be the case, at least for the rabbit. Beta receptor blockade with practolol (4 mg/kg), or with propranolol is without effect in preventing cardiac injury by norepinephrine [5]. This is consistent with findings in the present study where practolol also failed to inhibit lesion formation following tyramine (Fig. 6). On the other hand, alpha adrenoceptor blockade with phentolamine effectively eliminated myocardial injury in both studies. The increase in circulating catecholamines engendered by tyramine was for reasons which remain obscure significantly less in animals pretreated with phentolamine (Fig. 7). Nevertheless, average concentrations of 1520 ( + 171) pg/ml would be sufficient to cause significant myocardial injury, as indicated by the data in Figure 5. Thus it is most likely that blockade of myocardial alpha receptor-agonist binding was primarily responsible for the prevention of myofiber injury in this, as well as our previous study using NE. The use of phentolamine does not distinguish the alpha receptor sub-type, however. The conclusion that activation of alpha adrenoceptor pathways is an obligatory factor is further supported by the finding that identical myocardial injury patterns result from administration of the pure alpha agonist, methoxamine [5]. This also occurs in a dosedependent manner. While these results might not have been predicted on first consideration, there is growing evidence for species specific variation in myocardial alpha receptor sensitivity. Earlier studies have shown that methoxamine elicits significant positive inotropic responses in cat (21), rabbit (7), rat (19) and lamb (16), but not in dog hearts (2). This inconsistency is likely explained by the recent observations of Mukherjee et al. [18]. These workers found a markedly reduced number of alpha 1 receptors and increased number of beta receptors in dog heart sarcolemma compared with rat and rabbit. Binding properties were similar in all species. Thus the widely used dog model differs substantially in this regard from several other species in which myocardial alpha 1 receptor populations are probably substantially greater.
385
We have previously shown that pretreatment of rabbits with large doses of insulin (10 U/kg) substantially prevents the cardiomyopathic changes produced by NE [4, 32]. Insulin also sharply reduces contractility responses to NE in isolated cardiac muscle [17] and intact heart preparations [20]. Findings in the present study (Fig. 8, left panel) are consistent with these prior observations. Pretreatment with insulin significantly reduced the severity of myocardial damage from tyramine administration. The reduction was quantitatively identical with that seen in studies using NE [4, 32], further supporting the assumption that tyramine acts exclusively via catecholamine release. In view of the protective effects of exogenous insulin, we raised the possibility that insulin-deficient diabetics might be more prone to catecholamine cardiomyopathy [4]. Moreover, we have recently shown that inotropic sensitivity of diabetic hearts to alpha adrenoceptor stimulation is enhanced [6]. The present study failed to support this hypothesis, however. Using the lower dose of tyramine (200 ffg/min/kg), the average histological scores in controls and diabetic rabbits were identical (Fig. 8). This is consistent with our inability to demonstrate a greater propensity for NE-induced injury in diabetic rabbits (unpublished observations). It is also in agreement with the observations of Fein et al. [9] who used a streptozotocin diabetic rat model. Myocardial damage resulting from either isoproterenol or NE was identical with controls in this model as well. From these three lines of evidence it appears unlikely that insulin deficiency sufficient to cause moderate or severe [9] hyperglycemia increases myocyte vulnerability to catecholamine injury. It may be concluded from the present study that endogenous release of catecholamines is capable of inducing significant cardiomyopathic changes in the rabbit. Close positive correlations between the amount of tyramine given, resulting plasma catecholamine concentrations and the extent of lesion generation judged by histological analysis have been demonstrated. As was found with exogenously administered agonists, activation of alpha adrenergic pathways is the dominant mechanism in the rabbit model. Still to be resolved is whether myocyte injury is solely the conse-
386
S. Evans D o w n i n g and V. Chen
q u e n c e o f m e m b r a n e r e c e p t o r a c t i v a t i o n , or w h e t h e r i n d i r e c t effects of i n c r e a s e d a f t e r l o a d or c o r o n a r y v a s o c o n s t r i c t i o n [28] are c o n t r i b utory. A g o n i s t a c t i v a t i o n of a l p h a 1 receptors enhances phosphotidylinositol turnover with release of b o u n d i n t r a c e l l u l a r C a 2 +, as well as i n c r e a s e d u p t a k e o f e x t r a c e l l u l a r C a 2§ [8]. It is t e m p t i n g to s p e c u l a t e t h a t in excess this m a y trigger a c c e l e r a t e d d e g r a d a t i o n of m e m b r a n e p h o s p h o l i p i d s as d e s c r i b e d in i s c h e m i a [3-]. T h e l a t t e r is followed by m a r k e d increases in m y o c a r d i a l C a 2§ c o n c e n t r a t i o n a n d a m a n y - f o l d increase in passive C a 2+ perm e a b i l i t y of s a r c o p l a s m i c r e t i c u l u m . R e gardless, it is well established t h a t C a 2§
o v e r l o a d i n g is a f e a t u r e of c a t e c h o l a m i n e i n d u c e d m y o f i b e r i n j u r y [3, 10]. Precise deline a t i o n of m e c h a n i s m s a n d sequences m u s t await further investigation.
Acknowledgements T h e a u t h o r s are g r a t e f u l for the e x c e l l e n t t e c h n i c a l assistance p r o v i d e d by R o n a l d Gordon, William Stronk and Sandra Rancourt. D r C o l i n W h i t e p r o v i d e d v a l u a b l e h e l p w i t h statistical t r e a t m e n t of the data. S u p p o r t e d in p a r t by g r a n t s H L - 2 0 4 0 1 a n d H L - 0 8 6 5 9 f r o m the N a t i o n a l Institutes of Health.
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NUDEL, D. B., LE~, J. C., DOWNING, S. E. Reciprocal inhibition of cardiac responses to norepinephrine and insulin. AmJ Physio1233, H665 H669 (1978). RABINOWn'Z,B., CHUCK, L., KLIOERMAN, M., PARMLEY, W. W. Positive inotropic effects of methoxamine: evidence for alpha-adrenergic receptors in ventricular myocardium. A m J Physio1229, 582--585 (1975). REEVES,W. C., JACKSON, G. L., FLICKINOER, F. W., KWEE, H. G., SCHWITeR, E.J., WERNER, J., WHITSELL, L., BIDDLE, M. A., COPENHAVER, G., SHAIKH, B. S., ZELIS, R. Radionuclide imaging of experimental myocarditis. Circulation 63, 640-644 (1981). REICHENBACH, D. D., BEND1TT, E. P. Catecholamines and cardiomyopathy: the pathogenesis and potential importance ofmyofibrillary degeneration. Hum Pathol l, 125 150(1970). RONA,G., KAHNA, D. S., C:4APPEL, C. 1. Studies on infarct-like myocardial necrosis produced by isoproterenol: a review. Rev Can Bio122, 241-255 (1963). ROOKE,G. A., FEraL, E. O. Work as a correlate of canine left ventricular oxygen consumption, and the problem of catecholamine oxygen wasting. Circ Res 50, 273-286 (1982). SARNOFF,S. J., GILMORE,J. P., WEISFELDT,M. L., DAGGETT,W. M., MANSFIELD,P. B. Influence of norepinephrine on myocardial oxygen consumption under cotrolled hemodynamic conditions. Am J Cardiol 16, 217 226 (1965). SCHENK,E. A., Moss, A.J. Cardiovascular effect of sustained norepinephrine infusion. If. Morphology. Circ Res 18, 605-615 (1966). StMONS,M., DOWNING, S. E. Coronary vasoconstriction and catecholamine cardiornyopathy. Fed Proc 43, 423 (1984). SZAKACS,J. E., CANYON,A. l-Norepinephrine-myocarditis. Am J Clin Patho130, 425 434 (1958). VAN VLIET, P. D., BC'RCHELC,H. B., Trrus, J. L. Focal myocarditis associated with pheochromocytoma. N EnglJ Med 272, 1102-1108 (1966). WATKINS,D. B. Pheochromocytoma: A review of the literature.J Chronic Dis 6, 510 527 (1957). WERNER,J. C., LEE, J. C., DOWN1Na, S. E. Preservation of left ventricular function by insulin and experimental catecholamine cardiomyopathy. A m J Physio1238, H257 H262 (1980). ZAR,J. H. Biostatistical Analysis. W. D. McElroy, C. P. Swanson (Eds.), Englewood Cliffs, NJ: Prentice-Hall, Inc (1973).