Ultrastructural features of ethanol-induced cardiomyopathy in turkey poults

Camp. Biochem. Phnio!. Printed in Great Britain

Vol. 82A,

No. 4. pp. 939-943.

1985 8

0300-9629/85 $3.00 + 0.00 1985 Pergamon Press Ltd

ULTRASTRUCTURAL FEATURES OF ETHANOL-INDUCED CARDIOMYOPATHY IN TURKEY POULTS CAROLINE M. CZARNECKI,* STEPHEN W. SCHAFFER~ and ORAL A. EVANSON* *Department of Vet~rina~ Biology, College of Veterinary Medicine, University of Minnesota, St Paul, ME; 55108, USA. Telephone: (612) 376-4760; iDepartment of Pharmacology, College of Medicine, University of South Alabama. Mobile. AL 36688, USA. Telephone: (205) 460-6288 (Receitvd

17 April

1985)

Abstract-i. Alcoholic cardiomyopathy. characterized by cardiac hypertrophy, was induced in young turkey pouits with 57; ethanol. 2. Ultrastructural features included accumulation of glycogen, swollen mitochondria, myo~brillar lysis, increased number of lysosomes, diiated sarcoplasmic reticulum and dense myofibers. 3. Similarity of these alterations to those described in human alcoholic cardiomyopathy confirms the usefulness of the turkey poult as an animal model for this disease syndrome.

INTRODUCTION

The concept that a primary myocardial disease is associated with the chronic ingestion of alcohol became well established in the past 30 years. As a result, alcoholic cardiomyopathy is now a clinically recognizable form of heart disease (Brigden and Robinson, 1964; Ferrans, 1966; Burch and DePasquale, 1969; Burch and Giles, 1971; Regan, 1971; Regan and Haider, 198 1) and is considered to be one of the most frequent forms of cardiomyopathy (Parker, 1974). Contrary to early reports when the disease syndrome was commonly attributed to malnutrition (Bollinger, 1884; Aalsmeer and Wenckebach, 1929; Weiss and Wilkins, 1936; Blankenhorn, 1945), it has now been shown that alcoholic cardiomyopathy not only occurs in well-nourished individuals but is unresponsive to thiamine therapy (Butch and DePasquale, 1969). Experimental studies in various animal models and clinical observations of human patients support the premise that chronic alcoholism is the primary etiologic factor responsible for the development of the disease (Eliaser and Giansiracus, 1956; Brigden, 1957; Burch and Walsh, 1960; Tobin et al., 1967). There is considerable evidence to support the notion that alcohol is a direct myocardial toxin (Burch and DePasquale, 1969; Burch and Giles, 1971; Parker, 1974; Bing, 1978). Ingestion of alcohol in man is associated with prominent myocardial changes affecting oxidative enzymes (Burch and Giles, 1971; Regan, 1971; Parker, 1974; Bing, 1978; Edes et al., 1983), lipid metabolism (Burch and Giles, 1971; Parker, 1974; Bing, 1978) and morphologic features (Brigden and Robinson, 1964; Hibbs et al., 1965; Alexander, 1966; Burch and DePasquale, 1969; Burch and Giles, 1971; DeMakis et al., 1974; Parker, 1974; Steinberg and Hayden, 1981). Release of myocardial enzymes has been attributed to damage of the sarcolemma (Wendt et al., 1965). Reported morphologic alterations include flabbiness of the myocardium (Brigden and Robinson, 1964; Parker, 1974), dilatation and hypertrophy of all chambers (Brigden and Robinson, 1964; Burch and DePasquale, 1969; DeMakis et al., 1974; Parker, 1974), endocardial

fibrosis (Burch and DePasquale, 1969; Parker, 1974), loss of contractile elements (Hibbs et al., 1965; Alexander, 1966; Bulloch et al., 1972; Parker, 1974; Segel et al., 1975), deposition of lipid (Hibbs et al., 1965; Parker, 1974), swelling of the mitochondria (Hibbs et al., 1964; Alexander, 1966; Parker, 1974; Segal et al., 1975), dilatation of the sarcoplasmic reticulum (Hibbs ef al., 1965; Alexander, 1966; Bulloch et al., 1972; Segel et al., 1975) and an increase in glycogen (Hibbs et al., 1965; Alexander, 1966; Bulloch et al., 1972). The extent of these changes appear to be related to the severity and duration of the alcoholism (Segel et al., 1984). The study of alcoholic heart disease has been hampered by the inabiIity to reproduce consistently the condition in various animal models (Maines and Aldinger, 1967; Regan et al., 1974; Segel et al., 1975; Ettinger et al., 1976; Thomas et al., 1980; Kino et al., 1981; Friedman et al., 1982). Recently Noren et al. (1983) reported the use of the domestic turkey as a model of alcoholic congestive cardiomyopathy. Physiologic, mo~holo8ic and biochemical data were comparable to that reported in man with alcoholic cardiomyopathy. The purpose of this study is to document that the range in alteration of morphologic features at the ultrastructural level in alcohol-induced cardiomyopathy in turkeys is similar to that described in man from early to late stages of the disease. This is further evidence of the usefulness of the turkey as an animal model for studying the acute and chronic effects of alcohol on the human myocardium. MATERIALS AND

METHODS

Twenty one-day-old Broad Breasted White toms (Melof the Nicholas strain were obtained from a single hatch of commercial origin and placed randomly in two pens. Beginning at 1 day of age and continuing to 5 weeks of age, the poults in the experimental pen received 5% ethanol as their only Ruid source. Poults in the control pen received water ad lib. Body weights and ECG recordings were obtained at 2, 4 and 6 weeks of age. At 6 weeks of age, hearts from 2 control and 2 ethanol poults were perfused first with 0.1 M phos-

eugris gdopmo)

939

CAROLINE M. CZARNE~KI ef al

940

phate buffer and then with 3.00; buffered glutaraldehyde. Following fixation samples of tissue were removed from the free walls of the right and left ventricles and processed for electron microscopy. Tissues were post-fixed in 1% osmium tetroxide and 0.1 M phosphate buffer, dehydrated in a graded acetone series, embedded in epon araldite epoxy resin, sectioned on an LKB Ultrotome III, stained with uranyl acetate and lead citrate and photographed with a Zeiss IO electron photomicroscope at 60 KV. Hearts from the remaining I6 animals were excised, examined grossly for dilatation and weighed to determine heart to body weight ratios. Numerical data were analysed

using the Student’s l-test: P values jO.05 were considered to be significant.

RESULTS Alterations in the myocardial content of glycogen and lipid were consistent features in the ethanoltreated poults. In numerous myofibers, substantial amounts of glycogen were present among the mitochondria and myofibrils (Fig. I). In some instances the glycogen was associated with lipid droplets (Fig. 2). The latter were commonly seen in both normal and damaged fibers in the myocardium from ethanoltreated poults. Myofibrillar lysis was noted in many of the fibers from both ventricular walls (Figs l-5). The lysed fibers were observed adjacent to normal fibers (Figs I and 5) and, on occasion, a lysed myofibril was present next to a normal-appearing myofibril within the same myofiber (Fig. 3). Often the mitochondria in the lysed fibers appeared swollen (Figs 1-3). Although the size and shape of the mitochondria did not differ remarkably from that observed in control poults, an increase in the number of mitochondria was apparent in the myocardium of ethanol-treated poults. There was considerable variation in the alteration of other morphologic features in the myocardium from ethanol-treated poults. The intercellular space was often enlarged (Fig. 2). On occasion both the

Fig. 2. Lipid droplets (L) in partially Iysed fiber from wall of left ventricle. Note extensive intercellular space (arrows). X 12.000.

Fig. 3. Two myofibers from wall of right ventricle. Several myofibrils (MF) in upper fiber are partially lyscd. Edema (arrows) and swollen mitochondria (M) characterize lower fiber. x 12,000.

Fig. I. Partially lysed fiber from wall of left ventricle. Note glycogen aggregates (arrows) and swollen mitochondria (M). A normal fiber (NF) is present adjacent to lysed fiber. x 12.000.

Fig. 4. Numerous lysosomes (arrows) in partially lysed fiber from wall of left ventricle. x 12,000.

Ethanol-induced

cardiomyopathy

941

myofibrils. In one lysed fiber, the nuclear membrane had disappeared with the chromatin assuming a mitotic configuration (Fig. 5). Dense, contracted myofibrils were observed adjacent to normal myofibrils (Fig. 6). Mitochondria situated near these fibers were swollen and the sarcoplasmic reticulum was dilated. However, the mitochondria and sarcoplasmic reticulum associated with the adjacent relaxed myofibrils appeared to be normal. There were no significant differences in the mean body weights of control and ethanol-treated poults. However, the hearts from the ethanol-treated poults appeared to be flabbier and somewhat dilated. The mean weight of the heart ventricles from ethanoltreated poults was significantly (P < 0.01) greater than the mean weight of the heart ventricles from control poults (Table I). Likewise the heart to body weight ratio was statistically higher (P i 0.01) in ethanol-treated than in control poults. Fig. 5. Lipid droplets (L) and mitotic nucleus (N) in lysed fiber from wall of left ventricle. An adjacent normal fiber is present in upper right corner. x 10,000.

Fig. 6. Partially contracted dense myofibrils (arrows) adjacent to normal myofibrils from wall of left ventricle. Note dilated sarcoplasmic reticulum (SR) and swollen mitochondria (M) associated with the contracted myofibrils. x 12,000.

desmosomal attachments and the intercalated discs were disrupted. In some myofibers, the only alterations noted were edema and swollen mitochondria (Fig. 3). Lysosomes were commonly observed, being particularly numerous in lysed fibers (Fig. 4). Varying sizes of lysosomes were present both in a perinuclear position and among the more peripherally located

Table I. Mean body and heart weights (g) and heart to body weight ratios ,n control and ethanol turkey poults at 6 weeks of age Ethanol

Control Bad>

weight

(g)

Heart

weight

(g)

Heart

weight’

body ‘Mean

* 48.1’

5 3 i 0.0036

weight

(8)t

k 0.0001

of

:P

when

(8)

poulls. compared

with

1533.8

i

45.2

(8)

6.4 t 0.4 (8):

0.2 (8)

+ S.E.

tNumbrr

so.01

1499.5

controls.

0.0042

k 0.0002

(8)f

DISCUSSION

The development of a congestive cardiomyopathy due to chronic ethanol consumption is clearly recognized as a human medical problem (Ferrans, 1966; Regan, 1971; Regan and Haider, 1981). Less clear is its mechanism of development and histopathology. In a study of primary myocardial disease in 39 alcoholics, Tobin et al. (1967) identified three stages in the development of alcoholic cardiomyopathy. Stage 1 was defined as the phase prior to enlargement of the heart. Stage 2 was characterized by cardiac enlargement consistent with left ventricular hypertrophy. In stage 3 cardiac enlargement was associated with both left ventricular hypertrophy and dilatation. Cardiomegaly has been described as a consistent feature in chronic alcoholics (Brigden and Robinson, 1964; Burch and DePasquale. 1969; McDonald et al., 197 1; DeMakis et al., 1974). However, in the two animal models that have been used primarily to study the effect of alcohol ingestion on the development of congestive cardiomyopathy, prolonged exposure to ethanol has failed to produce the hypertrophy observed in humans (Bishop et al., 1967; Maines and Aldinger, 1967; Hall and Rowlands, 1970; Regan et al., 1974; Segel et al., 1975; Ettinger et ul., 1976; Thomas et al., 1980; Kino et ul., 1981). Recently Edes et al. (1983) and Noren et al. (1983) reported cardiomegaly in rats and turkey poults, respectively, following chronic ingestion of ethanol. The present study confirms the presence of cardiac hypertrophy and dilatation in turkey poults consuming approximately 23% of their total caloric intake as ethanol. Likewise. the increase in the ratio of heart to body weight has been reported in man (Steinberg and Hayden, 1981), turkeys (Noren et al., 1983) and rats fed a high concentration (25”/,) of ethanol (Segel et al., 1978). However, unlike the latter two studies, body weights of the ethanol-treated poults in this study were comparable to that of the controls. Thus malnutrition could be ruled out as a electropredisposing factor. Similarly, the cardiographic data indicated the absence of spontaneous round heart disease. These observations suggest that alcohol ingestion was primarily responsible for the cardiomyopathy observed.

CAROLINEM. CZARNECKIet

942

Manifestations of alcohol toxicity to the myocardium include decreased contractility (Maines and Aldinger, 1967; Lochner et al., 1969; Regan, 1971; Segel et al., 1975; Bing, 1978; Noren et al., 1983), increased tissue levels of glycogen (Hibbs et al., 1965; Alexander, 1966; Bulloch et al., 1972; Klein and Harmjanz, 1975) and triglyceride (Lieber et al., 1966; Regan et ul., 1974; Peuhkurinen et al., 1983) and numerous morphologic changes (Hibbs et al., 1965; Alexander, 1966; Burch and DePasquale, 1969; Burch and Giles, 1971; Bulloch et al., 1972; Parker, 1974; Klein and Harmjanz, 1975; Segel et al., 1975). Alterations which have been observed in experimental animals to date are neither comparable in severity nor extent to those seen in chronic alcoholics (Hall and Rowlands, 1970; Segel et al., 1975; Alexander et al.. 1977; Friedman et al., 1982). All the morphologic features reported in chronic alcoholics were observed in the turkey poults used in the present study. Several investigators have reported that the development of alcoholic cardiomyopathy involves early (acute) and later (chronic) changes (Segel et al., 1975; Alexander et al., 1977). In the acute phase of the disease, the primary alterations include derangement of the myofilaments and the mitochondria. The chronic phase is characterized by the deposition of lipid, development of interstitial fibrosis and presence of edema. In the turkey poult the acute and chronic stages can be produced in a relatively short time (5-6 weeks). This feature makes the turkey poult an acceptable animal model system for studying mechanisms involved in the development of the morphologic changes noted in chronic alcoholics where these alterations occur over a period of many years. The failure of reported animal models to produce the extent of myocardial changes noted in man has necessitated the development of new animal models. This urgency is compounded by the fact that some of the metabolic and morphologic abnormalities occurring prior to clinical manifestations may be reversed if recognized early enough. Although the relationship between the alcohol-induced metabolic changes and the development of cardiac lesions is unclear, there exists a need to identify early stages in the syndrome in an attempt to alleviate or prevent further myocardial damage. To this end the turkey poult emerges as an attractive animal model as it has been shown in this study and in a previous one (Noren et al., 1983) that the range of morphologic, mechanical and biochemical changes are comparable to those in man. Ac,lino\c/rd~ei)lent.v-The authors gratefully acknowledge the excellent care of poults provided by Carl Edborg, III, and Evelynn Eller. This study was supported by a grant from the Distilled Spirits Council of the United States. REFERENCES

Aalsmeer W. C. and Wenckebach K. F. (1929) Herz und Kreislauf bei der Beriberi Krankheit. Wien. ;. inn. Med. 16, 193-272. Alexander C. S. (1966) Idiopathic heart disease. Electron microscopic examination of myocardial biopsy specimens in alcohdlic heart disease. An;. J. Med. 41; i29-234. Alexander C. S.. Sekhri K. K. and Nagasawa H. T. (1977) Alcoholic cardiomyopathy in mice. Electron microscopic observations. .I. m&c. cell. Curdiol. 9, 247-254.

al

Bing R. J. (1978) Cardiac metabolism: alcoholic heart disease and myocardial

its contributions to failure. Circulation

58, 965-970.

Bishop M. B., Rosenblum I.. Davies J. N. P. and Stein A. A. (1967) Response of the rat myocardium to prolonged ethanol ingestion. Circ~ukrtion Suppl. II 3&69. Blankenhorn M. A. (1945) Diagnosis of beri-beri heart disease. Ann. intern. Med. 23, 398-404. Bollinger 0. (1884) Uber die Haufigkeit und Ursachen der idiopathischen Herzhypertrophie in Miinchen. Dr. rnc~/. W.&r. 10, 180-181. Brigden W. (1957) Uncommon myocardial diseases. Lancer 2, 117&1184, 1243-1249. Brigden W. and Robinson J. (1964)Alcoholic heart disease. Br. med. J. 2, 1283-1289. Bulloch R. T., Pearce M. B., Murphy M. L., Jenkins B. J. and Davis J. L. (1972) Myocardial lesions in idiopathic and alcoholic cardiomyopathy. Study by ventricular scptal biopsy. Am. J. Cardiol. 29, 15-25. Burch G. E. and DePasqualc N. P. (I 969) Alcoholic cardiomyopathy. Am. J. Cardiol. 23, 723-731. Burch G. E. and Giles T. D. (1971) Alcoholic cardiomyopathy. Concept of the disease and its treatment. Atn. J. Med. 50, 141-145. Burch G. E. and Walsh J. J. (1960) Cardiac insufficiency in chronic alcoholism. Am. J. Curdiol. 6, 865-874. DeMakis J. G., Proskey A., Rahimtoola S. H., Jamil M., Sutton G. C. Rosen K. M., Gunnar R. M. and Tobin J. R.. Jr. (1974) The natural course of alcoholic cardiomyopathy. Ann. intern. Med. 80, 293-291. Edes I., Ando, A.. Csanady M., Mazarean H. and Guba F. (1983) Enzyme activity changes in rat heart after chronic alcohol ingestion. Curdiocasc. Res. 17, 691-695. Eliaser M., Jr. and Giansiracus F. J. (1956) The heart and alcohol. Calif. Med. 84, 234-236. Ettinger P. O., Lyons M. M.. Oldewurtel H. A. and Regan T. J. (1976) Cardiac conduction abnormalities produced by chronic alcoholism. Am. Heart J. 91, 6678. Ferrans, V. J. (1966) Alcoholic cardiomyopathy. Am J. mrcl. Sci. 252, 89-104.

Friedman H. S.. Geller S. A. and Lieber C. S. (1982) The effect of alcohol on the heart. skeletal and smooth muscles. In Medicul Dkorders of’ Akoholi.~m : PLIIIIO genesis and Treumzent (Edited by Liebcr C. S.), pp. 436-479. W. B. Saunders, Philadelphia. Hall J. L. and Rowlands D. T.. Jr. (1970) Cardiotoxicity of alcohol. An electron microscopic study in the rat. An?. .I. Pothol.

60, 153-l 64.

Hibbs R. G.. Ferrans V. J.. Black W. C., Weilbaecher D. G.. Walsh J. J. and Burch G. E. (1965) Alcoholic cardiomyopathy: an electron microscope study. AP~I. HearI J. 69, 766- 179

Kino M.. Thorp K. A., Bing 0. H. and Abelmann W. H. (I 98 I ) Impaired myocardial performance and response to calcium in experimental alcoholic cardiomyopathy. J. molec. cell. Curdiol. 13, 981-989. Klein H. and Harmjanz D. (1975) Effect of ethanol infusion on the ultrastructure of human myocardium. Postgmd. med. J. 51, 325 327.

Lieber C. S.. Spritz N. and DeCarli L. M. (1966) Accumulation of triglycerides in heart and kidney after alcohol ingestion. J. c/in. Inrwr. (Abstr.) 45, 1041. Lochner A., Cowley R. and Brink A. J. (1969) Effect of ethanol on metabolism and function of perfused rat heart. An]. Heart J. 78, 770-780. Maines J. E. and Aldinger E. E. (1967) Myocardial depression accompanying chronic consumption of alcohol. Am. Hrrrrt

J. 73, 55-63.

McDonald C. D.. Burch G. E. and Walsh J. J. (1971) Alcoholic cardiomyopathy managed with prolonged bed rest. Ann. intern. Med. 74, 6X I-691 Noren G. R.. Staley N. A.. Einzig S., Mike11 F. L. and

Ethanol-induced Asinger R. W. (1983) Alcohol-induced congestive cardiomyopathy: an animal model. Curdiovasc. Res. 17, 81-87. Parker B. M. (1974) The effects ofethyl alcohol on the heart. J. Am. med. Ass. 228, 741-742. Peuhkurinen K. J., Kiviluoma K. T., Hiltunen J. K., Takala T. E. S. and Hassinen I. E. (1983) Effects of ethanol metabolites on intermediary metabolism in heart muscle. Pharmac. Biochem. Behac. 18 (Suppl.), 279-283. Regan T. J. (1971) Ethyl alcohol and the heart. Circularion 44, 957-963. Regan T. J. and Haider B. (1981) Ethanol abuse and heart disease. Circularion 64, Suppl. III, 14-19. Regan T. J., Khan M. I., Ettinger P. O., Haider B., Lyons M. M. and Oldewurtel H. A. (1974) Myocardial function and lipid metabolism in the chronic alcoholic animal. J. clin. Inuesr. 54, 740-752. Segel L. D., Klausner S. C., Gnadt J. T. H. and Amsterdam E. A. (1984) Alcohol and the heart. Med. Clins N.A. 68, 147-161. ’ Segel L. D., Rendig S. V., Choquet Y., Chacko K., Amsterdam E. A. and Mason D. T. (1975) Effects of chronic

cardiomyopathy

943

graded ethanol consumption on the metabolism, ultrastructure and mechanical function of the rat heart. Curdiovasc. Res. 9, 649-663. Steinberg J. D. and Hayden M. T. (1981) Prevalence of clinically occult cardiomyopathy in chronic alcoholism. Am. Heart J. 101, 461464. Thomas G., Haider B., Oldewurtel H. A., Lyons M. M., Yeh C. K. and Regan T. J. (1980) Progression of myocardial abnormalities in experimental alcoholism. Am. J. Cardiol. 46, 233-24 I. Tobin J. R., Jr., Driscoll J. F., Lim M. T., Sutton G. C., Szanto P. B. and Gunnar R. M. (1967) Primary myocardial disease and alcoholism. The clinical manifestations and course of the disease in a selected population of patients observed for three or more years, Circulation 35, 754-764. Weiss S. and Wilkins R. W. (1936) The nature of the cardiovascular disturbances in vitamin deficiency states. Trans. Ass. Am. Physns 51, 341-373. Wendt V. E., Wu C., Balcon R., Doty G. and Bing R. J. (1965) Haemodynamic and metabolic effects of chronic alcoholism in man. Am. J. Cardiol. 15, 175-184.