EXPERIMENTAL

AND

Ultrastructure Cor

MOLECULAR

of the

Pulmonale

28,

PATHOLOGY

Right

in the

( 1978)

107-118

Ventricle

Nonhuman

after Primate

Monocrotaline-Induced (Macaca

arctoides)’

TIMOTHY J. RACZNIAK, CHARLESF. CHESNEY,~AND JAMES R. ALLEN Department of Pathology, University of Wisconsin Medical Pathology Unit, Regional Primate Research Center, Madison, Wisconsin 53706 Received

April

29, 1977,

and in revised

School, University

and the Experimental of Wisconsin,

form August 1.5, 1977

The present study is concerned with the ultrastrnctural changes that occur in the right ventricle of Macaca arctoides monkeys following subcutaneous injection of the pyrrolizidine alkaloid monocrotaline. The monkeys showed right ventricular hypertrophy and congestive heart failure within 7 months following the initial injection of monocrotaline. At sacrifice, all of the monkeys had developed marked dilatation and hypertrophy of the right ventricle. Focal cytolysis involving myocytes, intracellular edema, and fibrosis were observed light microscopically. Ultrastrnctural changes of the right ventricle included hypertrophy and hyperplasia of the mitochondria, increase in intracellular matrical material, streaming and clumping of the Z-band material, disorganization and degeneration of myofibrils, dilatation of the sacroplasmic reticulum and transverse tubular system, increased number of free and attached ribosomes, and proliferation of intercellular collagen fibers.

INTRODUCTION Several reports have been published on the ultrastructure of pulmonary alterations induced by monocrotaline and its vasotoxic proximate metabolite, dehydromonocrotaline, during the last decade on tissues obtained from laboratory rats as well as monkeys (Merkow and Kleinerman, 1966; Valdivia et al., 1967; Kay et al., 1969; Butler, 1970; Chesney and Allen, 1973). However, there is no detailed report on the ultrastructural changes in the myocardium of monkeys that experienced monocrotaline-induced car pulmonale. In an earlier report, Allen and Chesney (1972) proposed that infant rhesus monkeys exposed to monocrotaline offered a unique noninvasive experimental model to study car pulmonale. Further clarification of the model in this report involves an evaluation of the ultrastructural changes in primate right ventricular myocardium with right ventricular hypertrophy and congestive heart failure.

MATERIALS

AND METHODS

Twelve laboratory-born infant Macacu arctoides sexes were evaluated. A 2% solution of monocrotaline

(stumptail) monkeys (S. P. Penick, New

of both Jersey)

1 This investigation was supported in part by United States Public Health Service Grants HL-10941 and RR-00167. Primate Center Publication No. 17-025. 2 Dr. Chesney is currently Associate Director, Toxicology Department, Hoechst-Roussel Pharmaceuticals Inc., Somerville, New Jersey 00876. 107 0014-4800/78/0281-0107$02.00/O All

Copyright 0 1978 by Academic Press, Inc. rights of reproduction in any form rewwd.

10s

RACZNIAK,

CHESNEY,

AND

TABLE Survival

Time

Animal

of Monkeys

I

Receiving

No.

ALLEN

Monocrotsline

Tolal

Injections

dose

Survival”

b&W

(days)

Experiment,nl N52F Q19M P66R1 Q42M NSF Q68F Q38F Q4iV Q57iV POYM

‘Jo 90 150 130 150 150 180 180 180 270

332 333 100 117 209 267 117 1% 267 280

Saline Snline

300 130

Cont~rol G31M ST285F u Experimental tory failnre.

animals

were

sacrificed

when

il was considered

t,hat, they

would

develop

respira-

was prepared according to the method of Hayashi et nl. (1967). At 1 month of age, a subcutaneous injection of monocrotaline (30 mg/kg body weight) was administered into the interscapular area of 10 monkeys. Two monkeys served as controls and received an injection of saline subcutaneously. Starting with the second month the experimental monkeys received either 30 or 60 mg of monocrotaline/kg body weight monthly. All animals were sacrificed at a time when cardiopulmonary dysfunction was manifested (Table I). At the time of sacrifice, the monkeys were anesthetized by intravenous injection of sodium pentobarbital (25 mg/kg). The th oracic cavity was opened immediately and several small samples of the right and left ventricle were taken for electron microscopy. The necropsy was then completed and sections of various organs were placed in 10% buffered formalin. These tissues were embedded in paraffin, sectioned at 5 pm, and stained with hematoxylin and eosin, and selected sections of the right and left ventricle, lungs, and liver were stained with Masson’s trichrome stain. Thin sections of the right and left ventricle were stained with uranyl acetate and lead citrate before electron microscopic evaluation. RESULTS The mean survival time of the 10 experimental monkeys depending on the dose of monocrotaline was 221 days, with a range between 100 and 355 days (Table I). There was a variation in the amount of time required to produce right ventricular hypertrophy and congestive heart failure, and this was dependent upon the dose. Hemogram and serum chemistry determinations of control and monocrotaline-injected animals are summarized in Table II. Experimental animals had a slight elevation of packed cell volume and hemoglobin concentration, and a decrease in total serum protein. At necropsy, there was right heart

MYOCARDIAL

ULTRASTRUCTURE

IN

COR

TABLE Hemogram

and Serum Chemistry Injected Subcutaneously

PULMONALE

IN

THE

PRIMATE

109

II Alterations in Nonhuman with Monocrotaline

Primates

Initial4

Terminalb

Experimenta& Hematocrit ( yO) Hemoglobin (g/100 ml) White count (103/mm”) Total serum protein (g/l00

ml)

42.2 13.3 10.1 6.3

f f f f

2.0 0.7 2.6 0.8

49.8 14.8 12.5 6.0

f 10.3 f 2.4 f 4.2 zk 1.2

ml)

39.0 13.0 11.0 6.4

f f f f

0.0 0.0 0.0 0.0

42.0 13.4 12.8 7.0

f f f f

Controlsd Hematocrit (yO) Hemoglobin (g/100 ml) White count (103/mmz) Total serum protein (g/100

1.4 0.5 0.7 0.2

0 Sample taken at 1 month of age and prior to monocrotaline injection. b Sample taken just prior to sacrifice. c Data expressed as mean value (&SD) of 10 monocrotaline-injected monkeys. d Mean values of two saline-injected controls.

and the right ventricular wall averaged 2.8 mm in thickness as compared to 1.5 mm in the controls (Table III). Right heart dilatation and hypertrophy were also typified by an increased circumference of the tricuspid and the right ventricular myocardium was pulmonary valve. Microscopically, edematous, the endocardium was moderately fibrotic, and there was considerable In addition, there were focal areas of hypertrophy of individual myocytes. myocytolysis associated with occluded small muscular arteries. The musculature of the left ventricle was normal except for a few enlarged myocytes. The vessels and endocardium of the left ventricle were unaffected. On microscopic examination, there was considerable hyalinization of the small muscular arteries and arterioles throughout the lung parenchyma. The affected vascular lumens were narrow or completely occluded. Associated with these vascular alterations were areas of connective tissue proliferation. dilatation,

Ultraotructwe

of Changes in the Right Ventricle

As was noted light microscopically, the myocytes were widely separated and there was a marked increase in the number of collagen fibers in the intercellular TABLE Right

III

Ventricular Thickness, and Tricuspid Valve and Pulmonary of Experimental and Control Monkeys at Postmortem Right ventricle thickness (mm)

Experimental” Controlb

Tricuspid circumference

2.8 1.5

a Mean value of seven experiment.al animals 6 Average value of two control animals.

Valve Circumference Examination valve (cm)

Pulmonary circumference

3.2 2.0 given

various

doses of monocrot.aline.

1.9 1.0

valve (cm)

110

RACZNIAK,

CHESNEY,

AND

ALLEN

FIG. 1. Note the increased number of mitochondria in the myocyte taken from the right ventricle of the heart of a monkey exposed to monocrotaline. Considerable variation exists in mitochondrial size and lucency of their matrical material (arrows). The arrangement of myofilaments is modified by the hyperplastic mitochondrin. Abundant ribosomes and short segments of sarcoplasmic reticulum (double arrow) are apparent in the sarcoplasm not occupied b!r other organelles. X10,900.

space. The changes that were present in the myocytes could be separated into two categories, i.e., those relating to hypertrophy and hyperplasia of cellular components and those associated with edema and degeneration. The majority of the cells was in the former category, where the most obvious change was the definite increase in the number of normal-appearing mitochondria. In many instances, these organelles comprised over one-half of the cell content. Although these hyperplastic mitochondria were located in their usual position between the myofibrils and around the nucleus, they also encroached on the spaces that other organelles usually occupied, thus distorting the normal cellular architecture (Fig. 1). The myofibrils in the majority of the cells were normal. However, due to the increase in mitochondria, their arrangement was less orderly with compression and shifting of their position within the sarcoplasm ( Fg. 1). The increased number of ribosomes and membranes of the sarcoplasmic

MYOCARDIAL

ULTRASTRUCTURE

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reticulum was apparent along the inner portion of the plasma membrane between the myofibrils, among the myofilaments, and around the nucleus. The majority of the ribosomes assumed a polysomal pattern and most frequently they were closely associated with short segments and vesicles of the sarcoplasmic reticulum and mitochondria (Fig. 1). Normal-appearing diads and triads formed by the sacroplasmic reticulum in the pIasma membrane were aIso apparent. In the myocytes that were undergoing degenerative changes, there was obvious intracellular edema. There were increased numbers of mitochondria and ribosomes as well as proliferated sarcoplasmic reticulum, and the organelles were widely separated (Fig. 2). There was considerable variation in mitochondrial size, matrical density, and closeness of contiguous cristae. In some cells, megamitochondria were dispersed among the normal-sized mitochondria (Fig. 3). Dilatation of the sarcoplasmic reticulum and accumulation of numerous small lipid dropIets were also apparent, Most severely altered were the myofibrils, with individual myofilaments being separated due to the increase in intracellular fluid (Fig. 4). There was also some disruption of filaments arrangment in the sarcomeres. Many of the filaments appeared to enter the Z band at various

FIG. 2. Intracellular edema is depicted in a right ventricular myocyte. Due to the edematous condition, only segments of the myofilaments are in the plane of section. Note the random distribution of the mitochondria and widely separated ribosomes. X9680.

112

RACZNIAK,

FIG. 3. In addition to mitochondria megamitochondria ( hl ) . X 6700.

CHESNEY,

hyperplasia,

AND

ALLEN

many

of the

affected

myocytes

contained

angles (Fig. 5). In other areas, several sarconicrcs were absent, leaving only matrical material between the Z bands. These changes were particularly obvious in the sarcomeres that abutted on intercalated discs (Fig. 6). There was also some streaming of the Z-band material. In many instances, the bands were much wider and less dense. Other Z bands extended from the sarcomeres along the inner surface of the plasma membranes and parallel to the myofibrils. There was some dilatation of the cisternae of the sarcoplasmic reticulum and enlargement of many of the segments of the transverse tubular system. In the more severely affected cells, there was also a separation of intercalated discs of contiguous cells. In the control animals the right ventricular myocardium showed mitochondria which were normal in size and shape. The sarcoplasmic reticulum consisted of a network of tubules of uniform distribution throughout the myofilaments. There was also uniform and parallel arrangement of the myofibrils. DISCUSSION The pyrrolizidine alkaloid monocrotaline was first incriminated as the cause of veno-occlusive disease by Bras and his associates in 1954 in the native pop-

MYOCARDIAL

ULTRASTRUCTURE

IN

COR

PULMONALE

IN

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ulation of the West Indies, where medicinal herb preparations containing this alkaloid were used to treat various illnesses (Bras et al., 1954). It was not until Lalich and Merkow (1961) ob served pulmonary vasculitis in rats fed monocrotaline that the cardiopulmonary aspects of this alkaloid began to be appreciated. Hayashi and Lalich (1967) observed marked right heart hypertrophy in rats with monocrotaline-induced pulmonary lesions. Allen et al. (1972) were able to show that the vascular lesions could be prevented by inhibiting the metabolism of monocrotaline. Butler et al. (1970) produced pulmonary vascular lesions with a pyrrole metabolite of monocrotaline. Hsu et a,?. (1975) demonstrated that the metabolites of monocrotaline bound readily to nucleic acids and protein in vivo and in vitro. It was also demonstrated in autoradiographic experiments that the monocrotaline metabolites were heavily localized in the pulmonary vasculature and could be correlated with the lesions alkaloids become toxic that were present (Hsu et al., 1976). The pyrrolizidine after their conversion to pyrrole derivatives by hepatic microsomal enzymes (Allen et al., 1972). Recent evidence by Hurley and Jago ( 1976) and Plestina

FIG. 4. As a result of the intracellular edema there was myofilaments in the fibrils. Note the various angles at which bands. x31,590.

considerable disruption the myofilaments enter

of the the 2

114

RACZNIAK,

CHESNEY,

AND

ALLEN

FIG. 5. The presence of wider Z bands and accumulation inner surface of the plasma membrane and parallel to the many of the affected myocytes. X14,925.

of Z-band material along myofihrils (arrow) occurred

the in

et al. (1977) has shown that the active metabolite of monocrotaline, dehydromonocrotaline, produces its effects in the first microcirculatory system it enters, and it is unlikely that monocrotaline or its metabolite dehydromonocrotaline has a direct toxic affect upon the myocardium, but rather the changes seen in the heart are secondary to changes present in the lung vasculature. The experimental data that have been reported from this laboratory indicate that monocrotaline and its vasotoxic metabolite dehydromonocrotaline are capable of producing sufficient vascular alterations in the lung to cause pulmonary heart disease in experimental animals. Allen and Chesney (1972) reported that infant rhesus monkeys given four injections of monocrotaline over a 6-month period developed an increase in hemoglobin, hematocrit, red cell mass, and arterial pC0, and a decrease in arterial ~0, and pH. There was also an increase in right heart and pulmonary artery pressures. At necropsy these animals displayed a marked enlargement of the right heart and extensive pulmonary vascular occlusion. Chesney and Allen (1973) reported that the pulmonary arteries and arterioles of monkeys exposed to monocrotaline had swollen

MYOCARDIAL

ULTRASTRUCTURE

IN

COR

PULMONALE

IN

THE

PRIMATE

11.5

endothelial eclls and hypcrtrophy of the medial musculature. In addition, fibrin and platelet thrombi within the intraalveolar capillaries were observed in rats given monocrotaline pyrrole (Lalich et al., 1977). There appears to be a relationship between the dose and the time sequence

cell

FIG. 6. Marked edematous adjacent to the intercalated

disruption of the sarcomere frequently disc. Note the paucity of myofilaments

occurred in areas of the in these areas. X8460.

116

RACZNIAK,

CHESNEY,

AND

ALLEN

in the dcvclopmcnt of car pulmouale after monocrotaliuc iiljcctiou. It has becu shown by varying the dose of monocrotaline pyrrole that the time sequence in the pathogenesis of cardiac hypertrophy and congestive heart failure may be controlled (Chesney et aE., 1974; Lalich et aE., 1977; Raczniak et al., 1977). Examination of the hearts of monkeys exposed to monocrotaline revealed hypertrophy of the myocytes in the right ventricle. In addition to the hypertrophy, the myocardium of the right ventricle was edematous, the myocytes were widely separated, and there was a proliferation of intercellular connective tissue. There was also considerable intracellular edema, as characterized by the abundance of organelle-free matrix in the myocytes. The ultrastructural modifications in the myocardium of the right ventricle were typical of the changes that arise as a result of hypertensive heart disease during cardiac hypertrophy and insufficiency (Maron et nl., 1975). The proliferation of mitochondria is one of the first compensatory changes that occurs in myocytes. It has been demonstrated that within a short period following aortic constriction there is an increased activity of mitochondrial enzymes in the ventricular myocardium and an enhanced uptake of mitochondrial protein precursors ( Aschenbrenner et al., 1972). There is also an increase in the synthesis of myosin and noncollagenous protein in the myocardial cells, which correlates well with the proliferation of myofilaments, ribosomes, and endoplasmic reticulum (Skosey et al., 1972). In addition, the proliferation of fibrous connective tissue is associated with an increase in DNA synthesis and uptake of collagen precursors (Grove et al., 1969; Skosey et al., 1972). Wh cn the hypertrophied myocardium is unable to compensate further, degenerative changes begiu to appear in the myocytes. In the presently reported study there were iutracellular edema, increased size of mitochondria, dilatation of the sarcoplasmic reticulum, and marked disruption of the myofilaments. There were also dilatation of the transverse tubular system and an occasional separation of the intercalated discs of contiguous cells. The above changes appear to be primarily related to the edema condition of the affected myocytes. As the heart musculature becomes hypoxic a decrease in the energy supply for normal cell function occurs (Pool and Braunwald, 1968). Altered myocyte permeability in heart failure has been related to decreased function of the Na+-K+ pump (Trump et al., 1971). Altered mitochondrial function associated with heart failure has been demonstrated in hearts in congestive failure following exposure to monocrotaline pyrrole. Mitochondrial electron transport and phosphorylating efficiency were shown to be markedly impaired ( Raczniak et al., 1977). With the availability of such a model the opportunity exists to more thoroughly evaluate by parallel biochemical, hemodynamic, cytochemical, and ultrastructural investigation what relationships exist between these variables and the subsequent development of experimental right ventricular hypertrophy and congestive heart failure. ACKNOWLEDGMENTS The authors gratefully thank Professor Joseph J. Lalich script. Appreciation is also expressed to Ms. J. Scheffler assistance and to Mr. R. Dodsworth for photography.

for his critical review and Ms. L. Carstens

of the man”for excellent

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REFERENCES ALLEN, J. R., and CHESNEY, C. F. (1972). Effect of age on development of car pulmonale in nonhuman primates following pyrrolizidine alkaloid intoxication. Exp. Mol. Puthol. 17, 220-23”. ALLEN, J. R., CHESNEY, C. F., and FRAZEE, W. J. (1972). Modifications of pyrrolizidine alkaloid intoxication in altered hepatic microsomal enzymes. Toxicol. Appl. Pharmacol. 23, 470479. ASCHENBRESNER, V., ALKIN, R., ZAK, R., NAIR, K. G., and RABINOWITZ, M. (1972). Increased turnover of mitochondrial constituents in cardiac hypertrophy and acute hypoxia in rats. In “Recent Advances in Studies on Cardiac Structure and Metabolism, Vol. 1, Myocardiology” (E. Bajusz and G. Rona, eds.), pp. 178-184. University Park Press, Baltimore. BRAS, G., JELLIFFE, D. B., and STUART, K. L. ( 1954). Veno-occlusive disease of the liver with nonportal type of cirrhosis occurring in Jamaica. Arch. Putlrol. 57, 285-300. BUTLER, W. W. (1970). An ultrastructural study of the pulmonary lesions induced by pyrrole derivatives of the pyrrolizidine alkaloids. J. Puthol. 102, 15-19. CHESNEY, C. F., and ALLEN, J. R. ( 1973). Monocrotaline induced pulmonary vascular lesions in nonhuman primates. Cardiocasc. Res. 7, 508-518. CI-IESNEY, C. F., ALLEN, J. R., and Hsu, I. C. (1974). Right ventricular hypertrophy in monocrotaline pyrrole treated rats. Exp. Mol. PuthoZ. 20, 257-268. GROVE, D., NAIR, K. G., and ZAK, R. ( 1969). Biochemical correlates of hypertrophy, III. Changes in DNA content, the relative contributions of polyploidy and mitotic activity. Circ. Res. 25, 463471. HAYASIII, Y., HUSSA, J. F., and LALICH, J. J. (1967). Cor pulmonale in rats. Lab. Invest. 16, 875-881. HAYASHI, Y., and LALICH, J. J. (1967). R enal and pulmonary alterations induced in rats by a single injection of monocrotaline. Proc. Sot. Esp. BioZ. Med. 124, 392-396. Hsu, I. C., RORERTSON, K. A., SHU~IAKER, R. C., and ALLEN, J. R. (1975). Binding of tritrated dehydroretronecine to macromolecules. Res. Commun. Chem. Puthol. PharmacoZ. 11, 99-106. Hsu, I. C., ROBERTSON, K. A., and ALLEN, J. R. ( 1976). Tissue distribution, binding properties and lesions produced by dehydroretronecine in nonhuman primates. Chem. BioZ. Interact. 12, 19-28. HUI~LEY, J. V., and JAGO, M. V. (1976). Delayed and prolonged vascular leakage in inflammation: The effects of dehydromonocrotaline on blood vessels in the rate cremaster. Puthology 8, 7-20. KAY, J. M., SMITH, P., and HEATH, D. ( 1969). Electron microscopy of CrotaZariu pulmonary hypertension. Thorax 24, 551-526. LALICH, J. J., and MERKOW, L. ( 1961). P u 1monary arteritis produced in rats by feeding Crotalaria spcctabilis. Lab. Incest. 10, 774-750. LALICH, J. J., JOHNSON, W. D., RACzNIAK, T. J., and SHUA~AKER, R. C. ( 1977). Fibrin thrombosis in monocrotaline pyrrole induced car pulmonale in rats. Arch. Puthol. Lab. Med. 101, 69-73. MARON, B. J., FERRAXS, V. J., and ROBERTS, W. C. (1975). Ultrastructural feature of degenerated cardiac muscle in patients with cardiac hypertrophy. Amer. J. Puthol. 79, 387-413. MERICOW, L., and KLEINERhlAN, J. ( 1966). microscopic study of pulmonary A n electron vasculitis induced by monocrotaline. Lab. Incest. 15, 547-564. PLESTINA, R., STONER, H. B., JONES, G., BUTLER, W. H., and MATTOCKS, A. R. (1977). Vascular changes in the lungs of rats after the intravenous injection of pyrrole carbamates. J. Pathol. 121, 9-18. POOL, P. E., and BRAUNWALD, E. (1968). Fundamental mechanisms of heart failure. Amer. J. Cardiol. 22, 7-15. RACZNIAK, T. J., CHESNEY, C. F., and ALLEN, J. R. ( 1977). Oxidative phosphorylation and respiration by mitochondria from normal, hypertrophied, and failing rat hearts. J. Mol. Cell. Cardiol. 9, 215-223. SKOSEY, J. L., ASCHENBRESNER, V., ZAK, R., and RABINOWITZ, M. (1972). Synthesis of colnoncollagen protein, and DNA during experimental myocardial hypertrophy lagen, myosin,

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B. F., CROKER, B. P., and MERGNEPi, W. J. ( 1971). The role of energy metabolism, and water shifts in the pathogenesis of cell injury. In “Cell Membranes: Biological Pathological Aspects” (G. W. Richter and D. G. Scarpelli, eds.; pp. 84-123. Williams Wilkins, Baltimore. VALDIVIA, E., LALICH, J. J., HAYASHI, Y., and SONNAD, J. (1967). Alterations in pulmonary alveoli after a single injection of monocrotaline. Arch. Pathol. 84, 64-76. TRUMP,

ion, and and

Ultrastructure of the right ventricle after monocrotaline-induced cor pulmonale in the nonhuman primate (Macaca arctoides).

EXPERIMENTAL AND Ultrastructure Cor MOLECULAR of the Pulmonale 28, PATHOLOGY Right in the ( 1978) 107-118 Ventricle Nonhuman after Prima...
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