231
Atherosclerosis, 32 (1979) 231-251 Scientific Publishers, 0 Elsevier/North-Holland
Ltd.
EFFECT OF CCLJNDUCED CIRRHOSIS ON THE PATHOPHYSIOLOGIC COURSE OF ACUTE MYOCARDIAL INFARCTION IN NONARTERIOSCLEROTIC VS ARTERIOSCLEROTIC MALE RATS
BERNARD
C. WEXLER and BRUCE P. GREENBERG
May Institute for Medical Research of the Jewish Hospital and the Departments of Medicine, Pharmacology, and Pathology, University of Cincinnati College of Medicine, Cincinnati, OH 45229 (U.S.A.) (Received 15 June, 1978) (Revised, received 26 October, (Accepted 6 November, 1978)
1978)
Summary Arteriosclerotic and nonarteriosclerotic rats were treated with carbon tetrachloride (CC&) to induce cirrhosis of the liver. Massive myocardial infarction was then induced in intact and Ccl,-treated animals. During acute necrosis (Days 1 thru 3), animals were killed at 4, 812 and 24 h on Days 1 and 2, and during myocardial repair on Days 4,5 and 8. During the induction of cirrhosis, animals developed polydypsia, polyuria, and hyperglycemia; during myocardial infarction, the arteriosclerotic + cirrhotic animals developed severe and persistent congestive heart failure, i.e., hydrothorax. Adrenal and thymus gland weights and corticosterone levels indicated that cirrhosis per se increased pituitaryadrenal activity, particularly in arteriosclerotic animals. Enzyme levels of SGOT and SGPT demonstrated severe hepatic damage due to cirrhosis and acute myocardial infarction. Blood triglycerides and cholesterol responded abnormally in cirrhotic animals during acute myocardial ischemia due to their entrapment within hepatic cells. The cirrhotic animals manifested poor myocardial repair with persistent foci of necrosis, calcification, and a high incidence of large, occlusive, atrial thrombi. It is suggested that cirrhosis interferes with lipid metabolism and adrenal steroid conjugation leading to abnormal levels of mineralocorticoids which favor congestive heart failure, poor myocardial repair, and atrial thrombosis. This work was supported in part by grants from the National Institute of Heart, Lung and Blood Diseases (HL-21418). the National Institute on Aging (AG-585). and the Southwestern Ohio Heart Association. Please address all correspondence to: Dr. Bernard C. Wexler. May Institute for Medical Research, 421 Ridgeway Avenue, Cincinnati, OH 45229, U.S.A.
232
Key words:
BUN - Cholesterol - Corticosterone - CPK - Free fatty acids - Glucose Hydrothorax - Isoproterenol - LDH - SGOT - SGPT - Triglycerides
Introduction During an acute myocardial infarction in rats, there is a transient but intense fatty infiltration of the liver [l-5]. The temporal appearance and severity of this fatty liver condition coincides with and parallels the intensity and duration of acute myocardial ischemia and necrosis. Concomitant with the development of the fatty liver condition, there is equally severe hyperlipidemia, e.g., triglycerides, free fatty acids, etc., accompanied by acute disappearance of peripheral adipose tissue sites, e.g., periadrenal, mesenteric, and carcass fat. During the subsequent myocardial repair phase, the fatty liver condition becomes resolved. Coincident with the fatty liver condition, the animals develop congestive heart failure evidenced by the accumulation of thoracic fluid, i.e., hydrothorax. The severity of the hydrothorax, i.e., quantitatively measured by aspiration, parallels the severity of the hepatic lipidosis. During this same period of hepatic lipidosis, congestive heart failure and myocardial necrosis, we have found a significant increase in aldosterone production [l]. We believe that the increased aldosterone production is mediated by impaired hepatic steroid conjugation due to the severe fatty infiltration of the liver. The increased aldosterone production is responsible, in turn, for the intense thoracic fluid accumulation. In an earlier report, we found that rats which were deliberately made to develop a fatty liver by feeding them erotic acid, all succumbed rapidly due to severe and unrelenting congestive heart failure when subjected to an acute myocardial infarction [ 61. These animals also manifested unusually severe myocardial infarcts and unusually high excursion of their serum enzymes (SGOT, SGPT and LDH), lipids, glucose, and BUN levels. In a related animal model, we have found that repeatedly bred male and female rats will become increasingly adipose, hyperlipidemic, and will develop severe fatty infiltration of the liver commensurate with the number and frequency of their breedings [7-111. In addition, these breeder rats spontaneously develop a Cushingoid spectrum of degenerative conditions, i.e., hyperglycemia, hypertension, arteriosclerosis, and premature aging. Paradoxically, when these arteriosclerotic breeder rats are subjected to an isoproterenolinduced myocardial infarct, they survive in greater numbers and show less excursion in serum enzymes, lipids, glucose, BUN, and corticosterone, than We believe the superior virgin rats which are free of vascular disease [l-5]. survival of the arteriosclerotic breeder rats is due to their pre-existing arterial disease, promoting growth of extra coronary artery collateralization and an altered endocrine response, i.e., the hypothalamic-pituitaryadrenal axis, to the stress of acute myocardial infarction. Carbon tetrachloride (CCL,) will induce cirrhosis of the liver in rats, inducing a state of functional hepatectomy [ 121. We exposed nonarteriosclerotic virgin males with normal livers, and arteriosclerotic breeder males with fatty infiltration of the liver, to chronic treatment with CC+ When these animals had
233
developed advanced cirrhosis of the liver, we subjected them to an acute isoproterenol-induced myocardial infarction. Our purpose was to determine whether cirrhosis of the liver would alter the usual pathophysiologic response to acute myocardial infarction, e.g., because of altered lipid, enzyme, or steroid metabolism which would accompany the cirrhotic condition, and whether the presence or absence of pre-existent arteriosclerosis, i.e., breeder rats, would affect the overall response to cirrhosis. Materials and Methods Adult (6 month old) Sprague-Dawley, male, virgin rats free of arterial disease and breeder rats with arteriosclerosis were used. One group of nonarteriosclerotic rats (n = 250) and arteriosclerotic rats (n = 250) were fed a regular commerical rat chow (Rockland: Teklad) which has a relatively low fat content (4%). This low fat diet will not produce a fatty liver or any suggestion of hyperlipidemia. Arteriosclerotic breeder rats are obese and develop hyperlipidemia and a mildly fatty liver spontaneously [l-5]. To induce cirrhosis of the liver, a second group of nonarteriosclerotic rats (n = 300) and arteriosclerotic rats (n = 300) were fed the same regular diet but were given 0.5 ml of CC& suspended in olive oil, 1 : 1 (v/v)/100 g b.w., subcutaneously, twice weekly for 9 weeks. (This large number of rats was used because prior experience demonstrated that not many rats with fatty livers will survive an isoproterenol-induced myocardial infarction [6]). All of the animals were housed in the same Animal Research Colony where temperature, humidity, and light were controlled carefully. AI1 of the animals were provided with food and water on an ad libitum basis. At several time intervals prior to the induction of myocardial infarction with isoproterenol, a number of animals given Ccl4 were autopsied to confirm the establishment of cirrhosis; some were injected with saline to ascertain whether the induction of cirrhosis had imparted any unusual responsiveness to the stress of injection - none was observed. The intact (regular diet), non arteriosclerotic and arteriosclerotic animals, and the cirrhotic, nonarteriosclerotic and arteriosclerotic rats were given two subcutaneous injections of isoproterenol spaced 24 h apart. The nonarteriosclerotic (virgin) rats were given 50 mg/lOO g b.w. Under normal conditions, the mortality rate due to isoproterenol-induced myocardial infarction at these dose levels is 50-60% in both nonarteriosclerotic and arteriosclerotic animals and most importantly, the area and severity of myocardium infarcted is equal in both types of animal. Despite the apparent discrepancy in dose level, the arteriosclerotic (breeder) rats received a larger total dose of isoproterenol because of their obesity. Isoproterenol causes more myocardial damage and is retained longer in obese animals. Animals (including controls, n = 24) were killed by instant decapitation following the first injection of isoproterenol at 4, 8, 12 and 24 h post-injection with a minimum of 8 animals per group, i.e., Day 1. On the following day, Day 2, all of the surviving animals were given the second injection of isoproterenol and the animals were killed at 4, 8, 12 and 24 h later. No further injections were given but animals from each of the groups were killed on Days 4, 5 and 8 after the second injection of isoproterenol.
234
At autopsy, blood was collected from the severed neck vessels from each animal, centrifuged in a refrigerated centrifuge and plasma stored in a deep freeze until time for analysis. The following biochemical parameters in each animal were analyzed using the Auto-analyzer (Technicon): creatine phosphokinase (CPK), transaminases glutamic oxaloacetic (SGOT) and glutamic pyruvic (SGPT), lactic dehydrogenase (LDH), triglycerides, free fatty acids, total cholesterol, glucose, and blood urea nitrogen (BUN). All of the above automated procedures are detailed in the manual published by the Technicon Co., “Automation in Analytical Chemistry”, Technicon, Mediad, Inc., New York. Circulating corticosterone (Cmpd.B) levels were measured by a fluorometric method of Guillemin et al. [13] as an index of adrenocortical secretory activity. Final body weight as well as the weight of pertinent organs were recorded. Gross observations were recorded of the heart and other salient pathological features. The heart and aorta, liver, adrenal, thymus, kidney were fixed in 10% buffered neutral formalin (Lillie) for histopathological analyses, i.e., paraffin and frozen sections. In addition to haematoxylin and eosin, special stains were used to demonstrate metachromasia and mucopolysaccharides (alcian and toluidine blue, Hale stain), elastic tissue (Verhoeff-van Gieson), calcium (von Kossa), and beta-cell granulation of the islets of Langerhans (aldehyde fuchsin Ponceau). Analysis of variance and bio-statistical analyses followed the procedures and tables cited by Snedecor [14]. Results General observations The animals tolerated the injections of CCL well. Only an occasional animal died due to the cirrhosis of the liver. Autopsies performed at various time intervals during the g-week CCL treatment demonstrated that cirrhosis developed promptly, within 7 days of treatment. The animals also developed polydypsia and polyuria and their urine tested positively for glucose and protein. Particularly striking was the fact that animals with cirrhosis were unusually sensitive to cutaneous stimuli, e.g., stroking of the fur, and were resistant to any handling. Within a few minutes of administration of the first injection of isoproterenol (Day l), all of the animals manifested marked tachycardia, rapid breathing, and prostration. Several hours later, the intact animals were moving about in their cages, eating and drinking as if they were normal, The CCl&reated animals remained prostrate although their tachycardia was somewhat lessened. Following the second injection of isoproterenol (Day 2), the severe tachycardia and prostration reappeared and were greatly exacerbated in cirrhotic animals. The usual mortality rate due to myocardial infarction was much greater in the cirrhotic animals, i.e., 56% of the intact, nonarteriosclerotic males vs 69% of the cirrhotic nonarteriosclerotic males, and 51% of the intact arteriosclerotic males vs 73% of the cirrhotic arteriosclerotic males. By Days 3 and 4, very few animals died and only 2 animals died during Days 5 through 8. Congestive heart failure, i.e., hydrothorax The severity of hydrothorax was determined
by aspirating
the thoracic
fluid
235
with a graduated syringe. Particularly striking was the unusually early and severe degree of congestive heart failure, i.e., hydrothorax, in the arteriosclerotic (breeder) rats with pre-existent fatty livers. Within 4-6 h of the first injection of isoproterenol, the thorax of animals with pre-existent fatty livers was inundated with fluid. Aspiration of this clear fluid demonstrated that within 4-6 h the animals with pre-existent fatty livers had already accumulated 9 ml (av.) of pulmonic fluid. This condition of congestive heart failure persisted unabated through the 3rd~-5th day after the second injection of isoproterenol. Contrariwise, the isoproterenol-treated animals with pre-existent normal livers and whose livers became fatty following isoproterenol injection (a consistent feature of isoproterenol-induced myocardial infarction), did not develop any overt signs of hydrothorax until the 2nd day of isoproterenol treatment. Further, their hydrothorax which averaged only 6 ml of thoracic fluid accumulation at its zenith, receded promptly and had resolved itself completely 24 h after the second injection of isoproterenol. (During the period of active thoracic fluid accumulation, animals are virtually anuric; during the period of thoracic fluid ebb, the animals manifest marked diuresis.) Concomitant with the disappearance of the hydrothorax condition in animals fed the regular diet, their livers showed complete resolution of the fatty infiltration. By direct contrast, all of the cirrhotic animals showed some persistence of the fatty liver condition and residual thoracic fluid throughout the experiment. The cirrhotic, arteriosclerotic (breeder) animals manifested collapsed and dehydrated intestines and at the zenith of their congestive heart failure, an accumulation of as much as 18 ml of thoracic fluid. Changes in organ and body weights The induction of cirrhosis caused little change in the body weight of nonarteriosclerotic rats (Table 1) but it had a marked catabolic effect in the arteriosclerotic animals (Table 2). Cirrhosis caused adrenocortical hyperplasia and increased adrenal weight concomitant with severe thymus gland involution in nonarteriosclerotic and arteriosclerotic animals (Tables 1 and 2); heart weights were considerably less in the cirrhotic animals (Tables 1 and 2); kidneys of the cirrhotic arteriosclerotic animals were reduced in size (cf. Tables 1 and 2). There were no notable changes in the weight of the testes of either the intact or cirrhotic animals. With the dynamic induction of acute myocardial damage by isoproterenol, the usual hour-by-hour, day-by-day fluctuations in body weight occurred with apparent equal intensity and direction in the intact and cirrhotic animals. The adrenal glands of the intact animals manifested the characteristic initial drop in weight concomitant with an increase in thymus gland weight during the early stages of myocardial infarction (Tables 1 and 2). By direct contrast, the cirrhotic animals manifested progressively increased adrenal weight and concomitant progressive involution of their thymus glands (Tables 1 and 2). The hearts of both the intact and cirrhotic rats, with or without arteriosclerosis, underwent a dynamic rise and fall in weight during the acute necrosis and repair phases of myocardial infarction (Tables 1 and 2). Similar, but less dynamic, changes were observed for the kidney weights.
348 f 15 8 3532 363 + 14 369 + 19
314+ 9 352 + 16 349 2 11 378 + 13
316f 7 385 + 10 371+ 12 366211
Isoproterenol, 2nd inj. 358f 4 4 h later 349? I 8 h later 337 f 13 12 h later 345 f 11 24 h later
Day 2
358 + 13
349 + io
359 + 11
363 +
Day 5
Day 8
26 + 1
29 + 2
31fZb
31+2b 3322’ 36f3a 3422”
+ f + f
12 11 a 21 a 23 a
35 27 24 a 31
170+
12a
187 f 31 a
34+
193 f 33 a
385 301 256 213
f + f t
a a a a 48 a 41 a 36 a 27a
19 31 47 61
1558 + 36 b 1513 + 37
80 ? 10 a 58 f 14 a
1607 k 41 a
? + ? f
107 + 84
1671 1723 1803 1927 1847 k 1831 + 1764 f 1718k
21 a 25 17 15
1469 + 40
Intact
(mg)
Heart wt
RATS
f f f +
61 67 59 77
a a a a
+ f + f
41 40 36 28
1406 f 29
1424 + 31
1476 + 31 a
1397 f 26 1408 + 28 1419 f 32 15312 41 a
1416 1408 1413 1403
1371 ? 32
Intact
(m&
f + f t
49 a 46 86 64
1335?
68
1284 + 53
1540 f 25 a
1464 ? 38 1400 ? 64 1410 f 50 14702 25b
1202 1353 1375 1367
1369 + 68
Cir.
OF ARTERIAL
Kidney wt
FREE
n = 8 rats each.
1403 f 22 a
1493 f 58 a
1818 f 63 a
1401 1426 1305 1689
1112 + 42 1182+41b 1260 + 56 a 12912 85’
1075 + 56
Cir.
SPRAGUE-DAWLEY
107 + 12 95 ?: 17 b 97 ?: 14 b 8 120f
104 f 113 f 129 + 1362
138 + 18
425 + 21 436 450 486 416
Cir.
Intact
38”2” 3
37+3a
41+2’ 40f3a 36+pb 36+ab
32f 3 3723a 41f3a 33 f 3
30 f 1
Cir.
Thymus wt (mzz)
MALE
Values are means i SE. Controls received no treatment, n = 24 rats; experimentals (Intact and cirrhotic) received isoproterenol, a P < 0.001, compared to control values on Day 0. bP < 0.05, compared to control values on Day 0.
9
368 + 15
8
351f
Day 4
2
23 + 1 22 f 2 27 + 2 30f4
242
360 f 18
11
Isoproterenol, 1st inj. 4 h later 8 h later 12 h later 24 h later
365+
Controls - No Rx
Intact
Day 1
Intact
Cir.
(6)
Day 0
Adrenal wt Cm&
Body wt.
Treatment
Day
COMPARISON OF BODY AND ORGAN WEIGHTS OF INTACT AND CIRRHOTIC, VIRGIN DISEASE AND SUBJECTED TO AN ISOPROTERENOL-INDUCED MYOCARDIAL INFARCTION
TABLE 1
2
33+2b 31 f 2
385 f 17
398 f 26
474 k
Day 8
82 + 11 113 f 13
108 f 14 a 106 f 13 a
32t
1
21 11 b 19 17
32 f 2
f f + +
66 ? 15
79 119 119 104
117 +_ 15a
35 26 a 41 a 20 b
39+2a
244 + 301 t 324 f 251? 69 f 11 131 f 14b 66f 7 78 f 10
1 3 4 1 161 + 18 a 146t ga 126f11a 125 f 12 a
+ + + f
36f2b 34 f 2 43+55a 45*5a
28 34 37 34
1202 f 1241 t 129lk 1413 +
44 87 39 a 60 a
1580 f 61 a 1535 k 90 a
1813 k 49 a 1712f37a
1596 f 71 a
a a a a
1892 f 51 a
42 51 61 62
18 60 41 71
1189 f 78 1437 + 97 a 1397 f 53 a 1600f74a + f + f
+ + f + a a a a
1721 1868 1919 1936
1569 1674 1661 1187
1129 f 44
CiL
? + ? f
a a a b
? f k f
78 81 a 59 67
1467 ?: 84 1407 + 93
1461 + 42 1399 t 26
1357 + 48
1434 1586 1347 1487
1271 ? 56 b 1569f68a 1516 + 56 a 1516+43a
1367 ? 57
CiI.
ARTERIOS-
29 31 26 29 b
31 42 27 31
1407 f 41
1407 f 1390? 1376 + 1321+
1531 1579 1498 1476
1409 f 28
Intact
(mg)
Kidney wt
RATS WITH EARLY
Values are means + SE. Controls received no treatment, n = 24 rats; experimentals (intact and cirrhotics) received isoproterenol. n = 8 rats each. a P < 0.001. compared to control values on Day 0. bP < 0.05. compared to control values on Day 0.
9
465 + 10
Day 5
34klb
338 -I:15
29 ? 2 33 + 3 37+2a 35f2a
459 f 13 b
f 13 + 16 + 17 +_13
Day 4
376 378 345 375
27 + 2 31 f 4 33 f 4 43+la
Isoproterenol, 2nd inj. 4862 7 4 h later 477 f 8 8 h later 465? 10 12 h later 461 f 9 24 h later
f 20 + 18 + 9 + 16
Day 2
379 394 393 372
1481 + 31
500f 11 533 ? 18 b 524+12b 499 f lib
11
Isoproterenol, 1st inj. 4 h later 8 h later 12 h later 24 h later
88f
Day 1
30 f 2
204 k 11
28 + 1
368 k 11
487 + 12
Controls - No Rx
Day 0
Intact
Intact
Intact
Cir.
Intact
Cir.
(mg)
(mi%) Cir.
Heart wt
Thymus wt
Adrenal wt (me)
Treatment
Day
(8)
SPRAGUE-DAWLEY
Body wt.
COMPARISON OF BODY AND ORGAN WEIGHTS OF INTACT AND CIRRHOTIC, MALE BREEDER CLEROSIS SUBJECTED TO AN ISOPROTERENOL-INDUCED MYOCARDIAL INFARCTION
TABLE
238
Blood chemistry Enzymes
Circulating creatine phosphokinase (CPK) levels rose sharply following the first challenging dose of isoproterenol with a blunting of the rise in CPK levels in the animals with cirrhosis. On Day 2, there was little or no change in circulating CPK levels in any of the animals following the second dose of isoproterenol. Both glutamic oxaloacetic and pyruvic .transaminases (SGOT and SGPT) were elevated abnormally prior to the induction of myocardial infarction in all of the animals having cirrhosis of the liver (Fig. 1). On Day 1 and particularly on Day 2, the SGOT and SGPT levels peaked at extraordinarily high levels but had receded to normal by Day 8 (Fig. 1). A similar dynamic rise and fall, with the incremental rise much higher in cirrhotic animals, was also observed in lactic dehydrogenase (LDH) levels. Lipids
Circulating triglyceride levels were reduced greatly in cirrhotic rats, particularly in the nonarteriosclerotic rats (Fig. 2). When challenged by isoproterenol on Days 1 and 2, the cirrhotic animals evinced very little change in circulating
teriosclerosis Intact Cirrhosis Arteriosclerosis Intact Cirrhosis P
$_ 300 r: % 250 4
Fig. 1. Changes in the circulating levels of glutamic pwuvic transsminase (SGPT) in intact and cirrhotic nonarteriosclerotic (virgin) and arteriosclerotic (breeder) male, Sprague-Dawley rats during the induction of an acute and massive myocardial infarction with the potent beta-adrenergic stimulating agent, isoproterenol. Some of the animals were treated with CC14 for 9 weeks to induce cirrhosis of the liver prior to the induction of myocardial infarction; the mtact animals had been raised on a low fat diet. Each point depicted is the mean f standard error; n = 24 for the controls, i.e.. intact and cirrhotic, prior to isoproterenol, n = 8 for the experimentals. i.e., intact and cirrhotic + isoproterenol. The same protocol applies to Figs. 2 through 6.
239 MU Virgins 0
- No Arteriosclerosis - Intact
l Breeders
- Cirrhosis - Early
Arteriosclerosis
A- intact A- Cirrhosis
MYOCARDIAL
NECROSIS
4 8 12 241
4 8 12
*
241
-HOURS-
DAYI
4
5
:* 81
DAYS
1
Fig. 2. Changes in circulating
DAY
I2
I
triglycerides.
triglyceride levels (Fig. 2). Although intact, arteriosclerotic (breeder) rats were hypertriglyceridemic prior to the induction of myocardial infarction, they showed a relatively modest increase during the acute cardiac necrosis phase, whereas the nonarteriosclerotic animals manifested a dynamic increase as well as a sustained hypertriglyceridemia (Fig. 2). The circulating free fatty acid levels rose and fell after each injection of isoproterenol but without any distinct pattern to differentiate intact from cirrhotic, or arteriosclerotic from nonarteriosclerotic rats. As in the case of triglycerides, the circulating cholesterol levels were depressed in the cirrhotic animals prior to treatment with isoproterenol (Fig. 3). Again, despite an initial marked hypercholesterolemia in the non-treated, intact, arteriosclerotic (breeder) rats, the intact, nonarteriosclerotic rats manifested the greatest increase in plasma cholesterol levels in response to each of the challenging doses of isoproterenol (Fig. 3). Glucose Although glucosuria was observed in all of the cirrhotic animals, only the nonarteriosclerotic (virgin) rats with cirrhosis were hyperglycemic (Fig. 4). During the induction of acute necrosis on Days 1 and 2, only the intact rats manifested the characteristic hyperglycemia which accompanies isoproterenolinduced myocardial infarction; the cirrhotic animals responded by becoming hypoglycemic (Fig. 4).
Protein The blood urea nitrogen
(BUN) was elevated
in cirrhotic
animals. During the
240 MnLE Virgins-No Arteriosclerosis 0 - ,n+oct
l-
400
Breeders
3x?
2 3oc ,
Cirrhosis - Early
A
- Intact
A
- Cirrhosis
Arteriosclerosis
-
2 L $ 25CI 5 2 2oc ,& 3
IX ,-
;: 3I$ IO
Atherosclerosis, 32 (1979) 231-251 Scientific Publishers, 0 Elsevier/North-Holland
Ltd.
EFFECT OF CCLJNDUCED CIRRHOSIS ON THE PATHOPHYSIOLOGIC COURSE OF ACUTE MYOCARDIAL INFARCTION IN NONARTERIOSCLEROTIC VS ARTERIOSCLEROTIC MALE RATS
BERNARD
C. WEXLER and BRUCE P. GREENBERG
May Institute for Medical Research of the Jewish Hospital and the Departments of Medicine, Pharmacology, and Pathology, University of Cincinnati College of Medicine, Cincinnati, OH 45229 (U.S.A.) (Received 15 June, 1978) (Revised, received 26 October, (Accepted 6 November, 1978)
1978)
Summary Arteriosclerotic and nonarteriosclerotic rats were treated with carbon tetrachloride (CC&) to induce cirrhosis of the liver. Massive myocardial infarction was then induced in intact and Ccl,-treated animals. During acute necrosis (Days 1 thru 3), animals were killed at 4, 812 and 24 h on Days 1 and 2, and during myocardial repair on Days 4,5 and 8. During the induction of cirrhosis, animals developed polydypsia, polyuria, and hyperglycemia; during myocardial infarction, the arteriosclerotic + cirrhotic animals developed severe and persistent congestive heart failure, i.e., hydrothorax. Adrenal and thymus gland weights and corticosterone levels indicated that cirrhosis per se increased pituitaryadrenal activity, particularly in arteriosclerotic animals. Enzyme levels of SGOT and SGPT demonstrated severe hepatic damage due to cirrhosis and acute myocardial infarction. Blood triglycerides and cholesterol responded abnormally in cirrhotic animals during acute myocardial ischemia due to their entrapment within hepatic cells. The cirrhotic animals manifested poor myocardial repair with persistent foci of necrosis, calcification, and a high incidence of large, occlusive, atrial thrombi. It is suggested that cirrhosis interferes with lipid metabolism and adrenal steroid conjugation leading to abnormal levels of mineralocorticoids which favor congestive heart failure, poor myocardial repair, and atrial thrombosis. This work was supported in part by grants from the National Institute of Heart, Lung and Blood Diseases (HL-21418). the National Institute on Aging (AG-585). and the Southwestern Ohio Heart Association. Please address all correspondence to: Dr. Bernard C. Wexler. May Institute for Medical Research, 421 Ridgeway Avenue, Cincinnati, OH 45229, U.S.A.
232
Key words:
BUN - Cholesterol - Corticosterone - CPK - Free fatty acids - Glucose Hydrothorax - Isoproterenol - LDH - SGOT - SGPT - Triglycerides
Introduction During an acute myocardial infarction in rats, there is a transient but intense fatty infiltration of the liver [l-5]. The temporal appearance and severity of this fatty liver condition coincides with and parallels the intensity and duration of acute myocardial ischemia and necrosis. Concomitant with the development of the fatty liver condition, there is equally severe hyperlipidemia, e.g., triglycerides, free fatty acids, etc., accompanied by acute disappearance of peripheral adipose tissue sites, e.g., periadrenal, mesenteric, and carcass fat. During the subsequent myocardial repair phase, the fatty liver condition becomes resolved. Coincident with the fatty liver condition, the animals develop congestive heart failure evidenced by the accumulation of thoracic fluid, i.e., hydrothorax. The severity of the hydrothorax, i.e., quantitatively measured by aspiration, parallels the severity of the hepatic lipidosis. During this same period of hepatic lipidosis, congestive heart failure and myocardial necrosis, we have found a significant increase in aldosterone production [l]. We believe that the increased aldosterone production is mediated by impaired hepatic steroid conjugation due to the severe fatty infiltration of the liver. The increased aldosterone production is responsible, in turn, for the intense thoracic fluid accumulation. In an earlier report, we found that rats which were deliberately made to develop a fatty liver by feeding them erotic acid, all succumbed rapidly due to severe and unrelenting congestive heart failure when subjected to an acute myocardial infarction [ 61. These animals also manifested unusually severe myocardial infarcts and unusually high excursion of their serum enzymes (SGOT, SGPT and LDH), lipids, glucose, and BUN levels. In a related animal model, we have found that repeatedly bred male and female rats will become increasingly adipose, hyperlipidemic, and will develop severe fatty infiltration of the liver commensurate with the number and frequency of their breedings [7-111. In addition, these breeder rats spontaneously develop a Cushingoid spectrum of degenerative conditions, i.e., hyperglycemia, hypertension, arteriosclerosis, and premature aging. Paradoxically, when these arteriosclerotic breeder rats are subjected to an isoproterenolinduced myocardial infarct, they survive in greater numbers and show less excursion in serum enzymes, lipids, glucose, BUN, and corticosterone, than We believe the superior virgin rats which are free of vascular disease [l-5]. survival of the arteriosclerotic breeder rats is due to their pre-existing arterial disease, promoting growth of extra coronary artery collateralization and an altered endocrine response, i.e., the hypothalamic-pituitaryadrenal axis, to the stress of acute myocardial infarction. Carbon tetrachloride (CCL,) will induce cirrhosis of the liver in rats, inducing a state of functional hepatectomy [ 121. We exposed nonarteriosclerotic virgin males with normal livers, and arteriosclerotic breeder males with fatty infiltration of the liver, to chronic treatment with CC+ When these animals had
233
developed advanced cirrhosis of the liver, we subjected them to an acute isoproterenol-induced myocardial infarction. Our purpose was to determine whether cirrhosis of the liver would alter the usual pathophysiologic response to acute myocardial infarction, e.g., because of altered lipid, enzyme, or steroid metabolism which would accompany the cirrhotic condition, and whether the presence or absence of pre-existent arteriosclerosis, i.e., breeder rats, would affect the overall response to cirrhosis. Materials and Methods Adult (6 month old) Sprague-Dawley, male, virgin rats free of arterial disease and breeder rats with arteriosclerosis were used. One group of nonarteriosclerotic rats (n = 250) and arteriosclerotic rats (n = 250) were fed a regular commerical rat chow (Rockland: Teklad) which has a relatively low fat content (4%). This low fat diet will not produce a fatty liver or any suggestion of hyperlipidemia. Arteriosclerotic breeder rats are obese and develop hyperlipidemia and a mildly fatty liver spontaneously [l-5]. To induce cirrhosis of the liver, a second group of nonarteriosclerotic rats (n = 300) and arteriosclerotic rats (n = 300) were fed the same regular diet but were given 0.5 ml of CC& suspended in olive oil, 1 : 1 (v/v)/100 g b.w., subcutaneously, twice weekly for 9 weeks. (This large number of rats was used because prior experience demonstrated that not many rats with fatty livers will survive an isoproterenol-induced myocardial infarction [6]). All of the animals were housed in the same Animal Research Colony where temperature, humidity, and light were controlled carefully. AI1 of the animals were provided with food and water on an ad libitum basis. At several time intervals prior to the induction of myocardial infarction with isoproterenol, a number of animals given Ccl4 were autopsied to confirm the establishment of cirrhosis; some were injected with saline to ascertain whether the induction of cirrhosis had imparted any unusual responsiveness to the stress of injection - none was observed. The intact (regular diet), non arteriosclerotic and arteriosclerotic animals, and the cirrhotic, nonarteriosclerotic and arteriosclerotic rats were given two subcutaneous injections of isoproterenol spaced 24 h apart. The nonarteriosclerotic (virgin) rats were given 50 mg/lOO g b.w. Under normal conditions, the mortality rate due to isoproterenol-induced myocardial infarction at these dose levels is 50-60% in both nonarteriosclerotic and arteriosclerotic animals and most importantly, the area and severity of myocardium infarcted is equal in both types of animal. Despite the apparent discrepancy in dose level, the arteriosclerotic (breeder) rats received a larger total dose of isoproterenol because of their obesity. Isoproterenol causes more myocardial damage and is retained longer in obese animals. Animals (including controls, n = 24) were killed by instant decapitation following the first injection of isoproterenol at 4, 8, 12 and 24 h post-injection with a minimum of 8 animals per group, i.e., Day 1. On the following day, Day 2, all of the surviving animals were given the second injection of isoproterenol and the animals were killed at 4, 8, 12 and 24 h later. No further injections were given but animals from each of the groups were killed on Days 4, 5 and 8 after the second injection of isoproterenol.
234
At autopsy, blood was collected from the severed neck vessels from each animal, centrifuged in a refrigerated centrifuge and plasma stored in a deep freeze until time for analysis. The following biochemical parameters in each animal were analyzed using the Auto-analyzer (Technicon): creatine phosphokinase (CPK), transaminases glutamic oxaloacetic (SGOT) and glutamic pyruvic (SGPT), lactic dehydrogenase (LDH), triglycerides, free fatty acids, total cholesterol, glucose, and blood urea nitrogen (BUN). All of the above automated procedures are detailed in the manual published by the Technicon Co., “Automation in Analytical Chemistry”, Technicon, Mediad, Inc., New York. Circulating corticosterone (Cmpd.B) levels were measured by a fluorometric method of Guillemin et al. [13] as an index of adrenocortical secretory activity. Final body weight as well as the weight of pertinent organs were recorded. Gross observations were recorded of the heart and other salient pathological features. The heart and aorta, liver, adrenal, thymus, kidney were fixed in 10% buffered neutral formalin (Lillie) for histopathological analyses, i.e., paraffin and frozen sections. In addition to haematoxylin and eosin, special stains were used to demonstrate metachromasia and mucopolysaccharides (alcian and toluidine blue, Hale stain), elastic tissue (Verhoeff-van Gieson), calcium (von Kossa), and beta-cell granulation of the islets of Langerhans (aldehyde fuchsin Ponceau). Analysis of variance and bio-statistical analyses followed the procedures and tables cited by Snedecor [14]. Results General observations The animals tolerated the injections of CCL well. Only an occasional animal died due to the cirrhosis of the liver. Autopsies performed at various time intervals during the g-week CCL treatment demonstrated that cirrhosis developed promptly, within 7 days of treatment. The animals also developed polydypsia and polyuria and their urine tested positively for glucose and protein. Particularly striking was the fact that animals with cirrhosis were unusually sensitive to cutaneous stimuli, e.g., stroking of the fur, and were resistant to any handling. Within a few minutes of administration of the first injection of isoproterenol (Day l), all of the animals manifested marked tachycardia, rapid breathing, and prostration. Several hours later, the intact animals were moving about in their cages, eating and drinking as if they were normal, The CCl&reated animals remained prostrate although their tachycardia was somewhat lessened. Following the second injection of isoproterenol (Day 2), the severe tachycardia and prostration reappeared and were greatly exacerbated in cirrhotic animals. The usual mortality rate due to myocardial infarction was much greater in the cirrhotic animals, i.e., 56% of the intact, nonarteriosclerotic males vs 69% of the cirrhotic nonarteriosclerotic males, and 51% of the intact arteriosclerotic males vs 73% of the cirrhotic arteriosclerotic males. By Days 3 and 4, very few animals died and only 2 animals died during Days 5 through 8. Congestive heart failure, i.e., hydrothorax The severity of hydrothorax was determined
by aspirating
the thoracic
fluid
235
with a graduated syringe. Particularly striking was the unusually early and severe degree of congestive heart failure, i.e., hydrothorax, in the arteriosclerotic (breeder) rats with pre-existent fatty livers. Within 4-6 h of the first injection of isoproterenol, the thorax of animals with pre-existent fatty livers was inundated with fluid. Aspiration of this clear fluid demonstrated that within 4-6 h the animals with pre-existent fatty livers had already accumulated 9 ml (av.) of pulmonic fluid. This condition of congestive heart failure persisted unabated through the 3rd~-5th day after the second injection of isoproterenol. Contrariwise, the isoproterenol-treated animals with pre-existent normal livers and whose livers became fatty following isoproterenol injection (a consistent feature of isoproterenol-induced myocardial infarction), did not develop any overt signs of hydrothorax until the 2nd day of isoproterenol treatment. Further, their hydrothorax which averaged only 6 ml of thoracic fluid accumulation at its zenith, receded promptly and had resolved itself completely 24 h after the second injection of isoproterenol. (During the period of active thoracic fluid accumulation, animals are virtually anuric; during the period of thoracic fluid ebb, the animals manifest marked diuresis.) Concomitant with the disappearance of the hydrothorax condition in animals fed the regular diet, their livers showed complete resolution of the fatty infiltration. By direct contrast, all of the cirrhotic animals showed some persistence of the fatty liver condition and residual thoracic fluid throughout the experiment. The cirrhotic, arteriosclerotic (breeder) animals manifested collapsed and dehydrated intestines and at the zenith of their congestive heart failure, an accumulation of as much as 18 ml of thoracic fluid. Changes in organ and body weights The induction of cirrhosis caused little change in the body weight of nonarteriosclerotic rats (Table 1) but it had a marked catabolic effect in the arteriosclerotic animals (Table 2). Cirrhosis caused adrenocortical hyperplasia and increased adrenal weight concomitant with severe thymus gland involution in nonarteriosclerotic and arteriosclerotic animals (Tables 1 and 2); heart weights were considerably less in the cirrhotic animals (Tables 1 and 2); kidneys of the cirrhotic arteriosclerotic animals were reduced in size (cf. Tables 1 and 2). There were no notable changes in the weight of the testes of either the intact or cirrhotic animals. With the dynamic induction of acute myocardial damage by isoproterenol, the usual hour-by-hour, day-by-day fluctuations in body weight occurred with apparent equal intensity and direction in the intact and cirrhotic animals. The adrenal glands of the intact animals manifested the characteristic initial drop in weight concomitant with an increase in thymus gland weight during the early stages of myocardial infarction (Tables 1 and 2). By direct contrast, the cirrhotic animals manifested progressively increased adrenal weight and concomitant progressive involution of their thymus glands (Tables 1 and 2). The hearts of both the intact and cirrhotic rats, with or without arteriosclerosis, underwent a dynamic rise and fall in weight during the acute necrosis and repair phases of myocardial infarction (Tables 1 and 2). Similar, but less dynamic, changes were observed for the kidney weights.
348 f 15 8 3532 363 + 14 369 + 19
314+ 9 352 + 16 349 2 11 378 + 13
316f 7 385 + 10 371+ 12 366211
Isoproterenol, 2nd inj. 358f 4 4 h later 349? I 8 h later 337 f 13 12 h later 345 f 11 24 h later
Day 2
358 + 13
349 + io
359 + 11
363 +
Day 5
Day 8
26 + 1
29 + 2
31fZb
31+2b 3322’ 36f3a 3422”
+ f + f
12 11 a 21 a 23 a
35 27 24 a 31
170+
12a
187 f 31 a
34+
193 f 33 a
385 301 256 213
f + f t
a a a a 48 a 41 a 36 a 27a
19 31 47 61
1558 + 36 b 1513 + 37
80 ? 10 a 58 f 14 a
1607 k 41 a
? + ? f
107 + 84
1671 1723 1803 1927 1847 k 1831 + 1764 f 1718k
21 a 25 17 15
1469 + 40
Intact
(mg)
Heart wt
RATS
f f f +
61 67 59 77
a a a a
+ f + f
41 40 36 28
1406 f 29
1424 + 31
1476 + 31 a
1397 f 26 1408 + 28 1419 f 32 15312 41 a
1416 1408 1413 1403
1371 ? 32
Intact
(m&
f + f t
49 a 46 86 64
1335?
68
1284 + 53
1540 f 25 a
1464 ? 38 1400 ? 64 1410 f 50 14702 25b
1202 1353 1375 1367
1369 + 68
Cir.
OF ARTERIAL
Kidney wt
FREE
n = 8 rats each.
1403 f 22 a
1493 f 58 a
1818 f 63 a
1401 1426 1305 1689
1112 + 42 1182+41b 1260 + 56 a 12912 85’
1075 + 56
Cir.
SPRAGUE-DAWLEY
107 + 12 95 ?: 17 b 97 ?: 14 b 8 120f
104 f 113 f 129 + 1362
138 + 18
425 + 21 436 450 486 416
Cir.
Intact
38”2” 3
37+3a
41+2’ 40f3a 36+pb 36+ab
32f 3 3723a 41f3a 33 f 3
30 f 1
Cir.
Thymus wt (mzz)
MALE
Values are means i SE. Controls received no treatment, n = 24 rats; experimentals (Intact and cirrhotic) received isoproterenol, a P < 0.001, compared to control values on Day 0. bP < 0.05, compared to control values on Day 0.
9
368 + 15
8
351f
Day 4
2
23 + 1 22 f 2 27 + 2 30f4
242
360 f 18
11
Isoproterenol, 1st inj. 4 h later 8 h later 12 h later 24 h later
365+
Controls - No Rx
Intact
Day 1
Intact
Cir.
(6)
Day 0
Adrenal wt Cm&
Body wt.
Treatment
Day
COMPARISON OF BODY AND ORGAN WEIGHTS OF INTACT AND CIRRHOTIC, VIRGIN DISEASE AND SUBJECTED TO AN ISOPROTERENOL-INDUCED MYOCARDIAL INFARCTION
TABLE 1
2
33+2b 31 f 2
385 f 17
398 f 26
474 k
Day 8
82 + 11 113 f 13
108 f 14 a 106 f 13 a
32t
1
21 11 b 19 17
32 f 2
f f + +
66 ? 15
79 119 119 104
117 +_ 15a
35 26 a 41 a 20 b
39+2a
244 + 301 t 324 f 251? 69 f 11 131 f 14b 66f 7 78 f 10
1 3 4 1 161 + 18 a 146t ga 126f11a 125 f 12 a
+ + + f
36f2b 34 f 2 43+55a 45*5a
28 34 37 34
1202 f 1241 t 129lk 1413 +
44 87 39 a 60 a
1580 f 61 a 1535 k 90 a
1813 k 49 a 1712f37a
1596 f 71 a
a a a a
1892 f 51 a
42 51 61 62
18 60 41 71
1189 f 78 1437 + 97 a 1397 f 53 a 1600f74a + f + f
+ + f + a a a a
1721 1868 1919 1936
1569 1674 1661 1187
1129 f 44
CiL
? + ? f
a a a b
? f k f
78 81 a 59 67
1467 ?: 84 1407 + 93
1461 + 42 1399 t 26
1357 + 48
1434 1586 1347 1487
1271 ? 56 b 1569f68a 1516 + 56 a 1516+43a
1367 ? 57
CiI.
ARTERIOS-
29 31 26 29 b
31 42 27 31
1407 f 41
1407 f 1390? 1376 + 1321+
1531 1579 1498 1476
1409 f 28
Intact
(mg)
Kidney wt
RATS WITH EARLY
Values are means + SE. Controls received no treatment, n = 24 rats; experimentals (intact and cirrhotics) received isoproterenol. n = 8 rats each. a P < 0.001. compared to control values on Day 0. bP < 0.05. compared to control values on Day 0.
9
465 + 10
Day 5
34klb
338 -I:15
29 ? 2 33 + 3 37+2a 35f2a
459 f 13 b
f 13 + 16 + 17 +_13
Day 4
376 378 345 375
27 + 2 31 f 4 33 f 4 43+la
Isoproterenol, 2nd inj. 4862 7 4 h later 477 f 8 8 h later 465? 10 12 h later 461 f 9 24 h later
f 20 + 18 + 9 + 16
Day 2
379 394 393 372
1481 + 31
500f 11 533 ? 18 b 524+12b 499 f lib
11
Isoproterenol, 1st inj. 4 h later 8 h later 12 h later 24 h later
88f
Day 1
30 f 2
204 k 11
28 + 1
368 k 11
487 + 12
Controls - No Rx
Day 0
Intact
Intact
Intact
Cir.
Intact
Cir.
(mg)
(mi%) Cir.
Heart wt
Thymus wt
Adrenal wt (me)
Treatment
Day
(8)
SPRAGUE-DAWLEY
Body wt.
COMPARISON OF BODY AND ORGAN WEIGHTS OF INTACT AND CIRRHOTIC, MALE BREEDER CLEROSIS SUBJECTED TO AN ISOPROTERENOL-INDUCED MYOCARDIAL INFARCTION
TABLE
238
Blood chemistry Enzymes
Circulating creatine phosphokinase (CPK) levels rose sharply following the first challenging dose of isoproterenol with a blunting of the rise in CPK levels in the animals with cirrhosis. On Day 2, there was little or no change in circulating CPK levels in any of the animals following the second dose of isoproterenol. Both glutamic oxaloacetic and pyruvic .transaminases (SGOT and SGPT) were elevated abnormally prior to the induction of myocardial infarction in all of the animals having cirrhosis of the liver (Fig. 1). On Day 1 and particularly on Day 2, the SGOT and SGPT levels peaked at extraordinarily high levels but had receded to normal by Day 8 (Fig. 1). A similar dynamic rise and fall, with the incremental rise much higher in cirrhotic animals, was also observed in lactic dehydrogenase (LDH) levels. Lipids
Circulating triglyceride levels were reduced greatly in cirrhotic rats, particularly in the nonarteriosclerotic rats (Fig. 2). When challenged by isoproterenol on Days 1 and 2, the cirrhotic animals evinced very little change in circulating
teriosclerosis Intact Cirrhosis Arteriosclerosis Intact Cirrhosis P
$_ 300 r: % 250 4
Fig. 1. Changes in the circulating levels of glutamic pwuvic transsminase (SGPT) in intact and cirrhotic nonarteriosclerotic (virgin) and arteriosclerotic (breeder) male, Sprague-Dawley rats during the induction of an acute and massive myocardial infarction with the potent beta-adrenergic stimulating agent, isoproterenol. Some of the animals were treated with CC14 for 9 weeks to induce cirrhosis of the liver prior to the induction of myocardial infarction; the mtact animals had been raised on a low fat diet. Each point depicted is the mean f standard error; n = 24 for the controls, i.e.. intact and cirrhotic, prior to isoproterenol, n = 8 for the experimentals. i.e., intact and cirrhotic + isoproterenol. The same protocol applies to Figs. 2 through 6.
239 MU Virgins 0
- No Arteriosclerosis - Intact
l Breeders
- Cirrhosis - Early
Arteriosclerosis
A- intact A- Cirrhosis
MYOCARDIAL
NECROSIS
4 8 12 241
4 8 12
*
241
-HOURS-
DAYI
4
5
:* 81
DAYS
1
Fig. 2. Changes in circulating
DAY
I2
I
triglycerides.
triglyceride levels (Fig. 2). Although intact, arteriosclerotic (breeder) rats were hypertriglyceridemic prior to the induction of myocardial infarction, they showed a relatively modest increase during the acute cardiac necrosis phase, whereas the nonarteriosclerotic animals manifested a dynamic increase as well as a sustained hypertriglyceridemia (Fig. 2). The circulating free fatty acid levels rose and fell after each injection of isoproterenol but without any distinct pattern to differentiate intact from cirrhotic, or arteriosclerotic from nonarteriosclerotic rats. As in the case of triglycerides, the circulating cholesterol levels were depressed in the cirrhotic animals prior to treatment with isoproterenol (Fig. 3). Again, despite an initial marked hypercholesterolemia in the non-treated, intact, arteriosclerotic (breeder) rats, the intact, nonarteriosclerotic rats manifested the greatest increase in plasma cholesterol levels in response to each of the challenging doses of isoproterenol (Fig. 3). Glucose Although glucosuria was observed in all of the cirrhotic animals, only the nonarteriosclerotic (virgin) rats with cirrhosis were hyperglycemic (Fig. 4). During the induction of acute necrosis on Days 1 and 2, only the intact rats manifested the characteristic hyperglycemia which accompanies isoproterenolinduced myocardial infarction; the cirrhotic animals responded by becoming hypoglycemic (Fig. 4).
Protein The blood urea nitrogen
(BUN) was elevated
in cirrhotic
animals. During the
240 MnLE Virgins-No Arteriosclerosis 0 - ,n+oct
l-
400
Breeders
3x?
2 3oc ,
Cirrhosis - Early
A
- Intact
A
- Cirrhosis
Arteriosclerosis
-
2 L $ 25CI 5 2 2oc ,& 3
IX ,-
;: 3I$ IO
