Basic Research in
Cardiology
Basic Res Cardio187:54-64
(1992)
Cardiac ultrastructural abnormalities in Syrian hamsters with spontaneous cardiomyopathy or subjected to cardiac overloads J. Perennec, M. Willemin, P. Pocholle, P. Y. Hatt, and B. Crozatier I.N.S.E.R.M. U2, H6pital Ldon Bernard, Limeil-Br6vannes, France Summary: Ultrastructural and morphometric abnormalities of Syrian hamster cardiomyopathy were compared to those observed in two different models of cardiac hypertrophy produced by mechanical overload (abdominal aortic stenosis, 60-day duration) or by isoproterenol injection during 15 days in normal Syrian hamsters of the same strain. Aspects of increased protein synthesis were observed in all three groups of animals. This was the only abnormality observed in the aortic stenosis group. Cardiomyopathy was different from the two other types of overload by the existence of large calcium deposits inside of the myocytes, by the presence of thin filaments and amorphous material accumulation suggesting abnormal synthesis and by a significant reduction of myofibrils at the heart-failure phase. Nuclear abnormalities with nuclear constrictions suggesting a division process and an increased number of myocytes with two nuclei were present in both spontaneous cardiomyopathy and isoproterenolinduced cardiopathy. Therefore, Syrian hamster cardiomyopathy appears to be different from cardiopathy induced by hemodynamic overload but, in spite of specific aspects, resembles that induced by isoproterenol injections, strengthening the hypothesis of a pathogenic role of catecholamines in the Syrian hamster cardiomyopathy. Key words: Syrian hamster; cardiomyopathy; nuclear abnormalities Introduction
Few reproducible models of cardiomyopathies are available in experimental animals. The most commonly utilized model is the cardiomyopathy of the Syrian hamster, which is a genetically determined disease with three stages of evolution: an ischemic phase followed by a compensated hypertrophy stage which terminates in congestive heart failure (4). A number of morphological studies of the Syrian hamster cardiomyopathy have already been published (3, 14, 19, 23, 34, 35, 37). However, no study determined which alterations were specific for cardiomyopathy and which alterations could be attributed to ischemia and to the hypertrophy process. The goal of our work was to analyze morphological alterations of the cardiomyopathic Syrian hamster in comparison with other cardiac alterations obtained in animals of the normal strain subjected to isoproterenol-indueed ischemia and to pressure-overload hypertrophy. The ultimate goal of our study was to attempt to determine the extent to which the genetically transmitted disease of the Syrian hamster simulated the observed forms of cardiopathies in patients (idiopathic cardiomyopathies or cardiac overloads due to hemodynamic alterations or to ischemic heart disease). Material and Method
1 Animals
Cardiomyopathic Syrian hamsters (UM-X 7.1 strain derived from Bio 14.6, Dr. Jasmin, Universit6 de Montr6al, Qu6bec, Canada) included 90-day-old animals (hypertrophy stage, n = 12) and 150-180707
Perennec et al., Syrian hamster cardiac ultrastructure
55
day-old animals (cardiac insufficiency stage, n = 14). They were compared to 20 healthy hamsters of the same age and of the same strain. An abdominal aortic constriction was performed in order to produce hypertrophy in another series of 13 healthy 45-day-old hamsters. The vessel was constricted above renal arteries with a plastic ring for producing a reduction of the aortic lumen by approximately 50-60 %. The animals were sacrificed 60 days after this constriction. Ten healthy animals of the same age served as controls for this series of animals. Cardiac hypertrophy was also produced in 15 healthy Syrian hamsters 60 days of age by daily subcutaneous injection of isoproterenol hydrochloride (0.5 mg/kg body weight) during 15 days. They were then sacrificed and compared to 10 controls of the same age. All animals (hypertrophied and cardiomyopathic) were sacrificed at the same age.
2 Fixation and tissue preparation Animals were anesthetized with intraperitoneal injection of pentothal (30 mg/kg of body weight). After cannulation of the abdominal aorta, heart arrest was induced in diastole by injection of procainamide in the IVC. Animals were retrogradedly perfused through the abdominal catheter with a fixation medium (1.25 % paraformaldehyde, 1.25 % glutaraldehyde buffered in cacodylate 50 mM with CaCla 4.1 raM; pH 7.4) for 5 rain under a perfusion pressure of 120 mmHg. After removal of the hearts from the thorax, transmural tissue samples were taken from the anterior, lateral, and posterior left-ventricular free walls. Tissue samples were immersed in the fixation medium, rinsed in cold sodium cacodylate buffer during 1 h, then postfixed in 2 % osmium tetroxyde for i h, and dehydrated in a graded ethanol series. Substitution of ethanol by propylene oxide was followed by incubation of the tissue in a mixture of propylene oxyde and epon. After 1 h the tissue was embedded in epon and polymerized for 24 h at 60 ~ Semi-thin sections were cut from each block, stained with blue toluidine and examined under a Zeiss microscope. Thin sections ( < 0.1 ~m) were prepared, mounted on uncovered copper grids, stained with uranyl acetate and lead citrate, and observed under a Zeiss EM 9 microscope.
3 Quantitative analysis Fiber diameter was determined using a micrometric ocular microscope. Longitudinal semi-thin sections were selected and examined at a magnification of x 500. Diameters were determined at the level of the nuclei; 250 measurements per sample were performed. Number of nuclei per cell was determined by examination of longitudinal semi-thin sections at a magnification of x 500; 250 myocytes per sample were examined. The fraction of cell volume occupied by mitochondria, myofibrils, and sarcoplasm was determined by the point-counting analysis described by Weibel (46), using a grid composed of intersecting lines 1-cm apart and placed upon an electron micrograph at a final magnification of • A minimum of 2000 points was counted per animaI. Volume fractions of subcellular components were calculated by dividing the number of points overlying a given component by the total points overlying the tissue. All data of different groups of animals were compared statistically using a one-way analysis of variance for multiple means. When a significant trend was found by the F test, Student's t-test was used to compare two different means with a correction by Bonferoni's method of multiple means. Results were considered for p < 0.05. Results
1 Morphological data W e o b s e r v e d lipid droplets inside myocytes in all groups, including the control group, w h e n animals were sacrificed in a u t u m n or in winter. Cardiomyopathic Syrian hamsters at the hypertrophy stage: No obvious myofibrillar alterations were seen in this group. F e a t u r e s evoking e n h a n c e d synthesis were c o m m o n : large nuclei with several nucleoli or p s e u d o vesicles, proliferative Golgi a p p a r a t u s , d e v e l o p e d sarcoplasmic reticulum, loci of m y o f i l a m e n t synthesis. A constriction of the middle p a r t of nuclei was a peculiar aspect o f t e n o b s e r v e d in this g r o u p of animals (Fig. 1).
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Basic Research in Cardiology, Vol. 87, No. I (1992)
Fig. t. Electronmicrograph of nuclei of cardiomyopathic Syrian hamster at the hypertrophy stage, showing a peculiar aspect of the nucleus with an identation in its middle part. This aspect was often encountered (~ 2/~of cells). Mag.: x 10 000.
Cardiomyopathic Syrian hamsters at the cardiac insufficiency stage: In this group a number of alterations was observed in myofibrils, intercalated discs, mitochondria, T tubules and nuclei. Disorganization of myofibrillar arrangement was sometimes seen. Different degrees of structural alterations were observed. Z bands were often irregular with disappearance of their usual alignment in adjacent myofibrils. Z band material spread along the sarcomere in the direction of adjacent myofibrils (Fig. 2). During this end stage, features of synthesis and degeneration coexisted. Myofibrils were scarce in large myocytes. Foci of amorphous material or of thin filaments evoked abnormal synthesis (Fig. 3). Intercalated discs were very irregular, sometimes duplicated. We observed disappearance of thin filaments inserted upon them. This disappearance was predominant on one side of the intercalated disc, and T tubules proliferated. They were often filled with dense material. Mitochondrial size varied from very small to giant mitochondria. Abnormalities of nuclei described in the hypertrophic group were more frequent at the end stage of the disease. Amorphous circular concretions of calcium deposits occupied an important part of some cells adjacent to cells appearing normal (Fig. 4). Normal hamsters with aortic stenosis: Only features of synthesis were observed. Particularly large mitochondria were frequently noted. Nuclei were enlarged, but without indentation aspects. Normal hamsters o f the isoproterenolgroup: Foci of fibrosis were developed. Morphological aspect was similar to that of the stenosis group. Indentations of nuclei similar to those observed in the heart-failure group were sometimes, but rarely encountered.
Perennec et al., Syrian hamster cardiac ultrastructure
57
Fig. 2. Electronmicrograph of a cardiomyopathic Syrian hamster at the cardiac insufficiency stage. Z-band material spread in the direction of the membrane (arrow). Note calcium deposits (Ca) and collagen fibers (C). Mag.: x 22 000.
2
Quantitative data
Myofiber diameter was significantly larger in the four pathologic groups as compared with the control group (all Ps < 0.001; Table 1). In both the control group and the stenosis group the majority of myocytes had one nucleus. In contrast, in cardiomyopathic Syrian hamsters there were significantly more myocardial cells with two nuclei than cells with one nucleus. In the isoproterenol group, an intermediate result was found (Fig. 5). The morphometric study showed (Table 1) a significantly decreased myofibrillar volume in the cardiomyopathic hamsters during the ultimate stage only. It was related to the loss of myofibrils and mitochondrial volume being unchanged. In the other groups, results were similar to the controls. Discussion
Some of the presently described alterations were common to all types of pathological processes (genetically transmitted disease and cardiomyopathy induced by isoproterenol or pressure overload). In contrast, some alterations were specific for genetically derived disorder or were common with isoproterenol induced cardiopathy.
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Basic Research in Cardiology, Vol. 87, No. 1 (1992)
Table 1. Morphometric data in Syrian hamsters.
Controls (n = 40) Cardiomyopathic (hypertrophy) (n = 12) Cardiomyopathic (cardiac insufficiency) (n = 14) Aortic stenosis (n = 13) Isoproterenol (n = 15) F
Myofiber diameters (~m)
V Myofibrils (%)
V Mitochondria
V Cytoplasm (%)
15.2 + 0.7
59.7 + 6.2
24.4 • 4.7
15.9 + 3.7
23.5 _+1.38*
57.1 _+7.8
23.9 + 5.6
18.9 _+4.8
23.4 _+ 1.7"
46.4 +_6.24
22.8 + 5.2
30.8 • 5.11
23.4 • 2.10
63.7 + 6.2
23.1 _+3.9
13.2 + 4.7
21.3 • 1.72"
57.7 • 5.1
23.1 _+3.8
19.2 • 4.3
p < 0.05
p < 0.001
NS
p < 0.001
Myofiber diameters and fractional volume occupied by myofibrils, mitochondria, and cytoplasm (mean • 1 s.d.) were compared in the five groups of Syrian hamsters (normal, cardiomyopathy at the compensated and heart failure stage and pressure and isoproterenol induced overload). *: p < 0.05; ~: p < 0.005 with the control group.
Cardiac alterations c o m m o n to all three types o f cardiopathies
The same degree of increase of cellular diameter was observed without modifications of the fractional volumes of mitochondria, myofibrils, and sarcoplasm in all three types of cardiopathies (Tables 1, 2) with features of increased protein synthesis similar to those described in mechanical overloads of other species (7, 15, 16). Larger values were found in control animals by Sorenson et al. (43). However, they used isolated cells which were not subjected to strains due to interstitial tissues. The increase in nucleus size is a commonly described pattern in a n u m b e r of clinical and experimental settings (13, 26, 39, 42). Similarly, a n u m b e r of alterations observed in myocardiopathic Syrian hamsters examined at the stage of heart failure were similar to those previousTy described in cardiopathies of other etiologies. Multiplication of intercalated discs was described in experimental and human cardiac overloads (1, 22, 25, 30) and had been attributed to the increased tension on cardiac cells due to the hemodynamic overload. Myofibrillar volume decrease is commonly observed in h u m a n heart failure due to hemodynamic overload (26). This pattern is in contrast with that described by Lazarus et al. (23) in the same experimental model as ours, but our results are in agreement with other descriptions of the hamster cardiomyopathy at the end-stage of heart failure (37). They can be compared to those of Venkatakrishnan et al. (44), who found an increased rate of myosin degradation with a loss of myosin. Degenerative aspects with myofibrillar lysis have also been described in h u m a n myocardium of patients with chronic aortic valve disease (31) and by Hart et al. (17) in rats subjected to pressure overload with aspects of disruption and clarification of intercalated discs. However, features of myofibrillar synthesis persisted in the cardiomyopathy of the Syrian hamster, even at this stage of heart failure. The association of aspects of synthesis and degeneration
Perennec et al., Syrian hamster cardiac ultrastructure
59
Fig. 3. Electronmicrograph of cardiomyopathic Syrian hamster at the cardiac insufficiency stage with loci of amorphous material (+) may be evoking abnormal synthesis or degraded material. This aspect was not encountered in controls. Mag.: • 10 000.
has been described in experimental cardiopathies induced by hemodynamic overload. Features evoking increased myofibrillar synthesis had been noted by Anversa et al. (2), Bishop et al. (5), Hatt et al. (18), and Legato (24) demonstrated an increased leucine incorporation in Z bands of rat hearts subjected to an aortic constriction. The spread of Zband material along the sarcomere (Fig. 4) is similar to that described by Hatt et al. (18) in rat pressure overload. It must be noted that all these experimental studies examined the myocardium at the compensated phase. In contrast with the persistence of features of myofibrillar synthesis in heart failure of the Syrian hamster, we (38) did not find such features in human cardiomyopathy studied at the end-stage of heart failure. In this respect, human cardiomyopathy is thus different from that of the Syrian hamster. Cardiac abnormalities specific to the inherited disease
A calcium accumulation in myocardial ceils was a specific feature of the cardiomyopathy of the Syrian hamster observed at the end-stage of heart failure (Figs. 4A and B). Although calcium accumulation has been described in myocardial cells in the early phase of Syrian hamster cardiomyopathy (32, 34) or in the connective tissue at the stage of heart failure (32), we did not find in the literature any description of intracellular calcium at this stage or large calcium concretions such as those found here (Figs. 4A and B). Intracellular calcium
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Basic Research in Cardiology, Vol. 87, No. I (1992)
Fig. 4. Electronmicrograph of cardiomyopathic Syrian hamster at the cardiac insufficiency stage. A) calcium deposits (Ca) were observed adjacent to a myocyte appearing normal (My). Mag. x 5000. B) amorphous circular concretions of calcium (+). Mag.: x 20000.
61
Perennec et al., Syrian hamster cardiac ultrastructure
overload has been considered as responsible for the initiation of myocardial lesions due to different reasons: sarcolemmal membrane permeability changes (29, 36, 45), increased sympathetic activity (6, 28, 29, 41) with an increased alpha-receptor number and response to norepinephrine (20), depression of the Na-K ATPase activity (29, 36), or an increased number of slow calcium channels (45). Cardiac abnormalities c o m m o n to inherited and isoproterenol-induced cardiomyopathies
Calcium accumulation was not observed in the isoproterenol-induced cardiopathy of the Syrian hamster, but a number of features were common to this type of cardiopathy and to the inherited disease. Thin filament accumulation suggesting a myofibrillar degradation was described both by Nag et al. (33) in cell cultures of cardiomyopathic Syrian hamster and in isoproterenol-induced ischemia in rats by Csapo et al. (11), who suggested a redistribution of actin. The number of cells with 2 nuclei was increased both in isoproterenol and inherited cardiopathy of the Syrian hamster as compared with normal hamsters in which 80 % of the cells had only one nucleus (Fig. 5). This species is thus different from other species in which cells with two nuclei predominate (21). This result is different from that of Campbell et al. (9) and of Nag et al. (33), who found a majority of ceils with two nuclei in cell cultures of normal Syrian hamsters. A possible explanation for this discrepancy is a selection of cells with two nuclei during cell isolation. Although such a selection has never been described, it is difficult to assess, but cannot be excluded. The most striking nuclear abnormality found in myocardiopathic Syrian hamsters was the aspect of nuclear indentation observed at the hypertrophy stage (Fig. 1), and even more frequently during the end stage of heart failure. It was never observed in the control group, and was not the result of a cellular contraction. The significance of this aspect is unknown. It can be explained by a cell fusion such as that shown
1OO
.[ L
Cells
with
1 nucleus
u z
-~
C e l l s w i t h 9, n u c l e i p < 0.01 with control
c~
g -r v-
50
g
CONTROL
A.S.
ISO.
H.C.M.
Fig. 5. Number of nuclei per cell. Bars indicate 1 sem. The percentage of cells with two nuclei was increased in the isoproterenol (ISO) and cardiomyopathy (HCM) groups as compared with the control group (* p < 0.01). The overall F value was highly significant (p < 0.001). A. S, = aortic stenosis group.
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Basic Research in Cardiology, Vol. 87, No. 1 (1992)
in young rats which leads to binuclear cells, to an acytokinetic mitosis similar to that found in the liver or to a polyteny, that is, a complete absence of mitotic mechanism leading to a duplication of DNA (8). This latter phenomenon would be in agreement with the findings of Paterson et al. (37) who found an increase of DNA which was larger than that of proteins without increases of endothelial or conjunctive cells in cardiomyopathic Syrian hamsters. Similarly, Liew et al. (27) found in the same disease an increased amount of histones, suggesting an increased gene activity. These nuclear aspects were also found in Syrian hamsters submitted to isoproterenol injections. A polyploidization was previously described in isoproterenoMnduced cardiopathies of rats (10, 11, 40). The similarity between nucleus abnormalities and thin filament accumulation found in cardiomyopathic Syrian hamsters and normal Syrian hamsters subjected to isoproterenol injections suggests that, as already proposed, the inherited disease of this species may be due to increased myocardial sympathetic activity with local elevation of norepinephrine concentration leading to loci of necrosis (12). In conclusion, it appears from our data that a number of electron microscopic aspects of the cardiomyopathy of the Syrian hamster (foci of amorphous material, nuclear abnormalities, calcium deposits) are different from those observed in hemodynamic overload of the same species. Nuclear abnormalities and thin-filament accumulation more resemble the aspects found in isoproterenoMnduced cardiomyopathy. The persistence of signs of protein synthesis in the Syrian hamster cardiomyopathy is different from its absence in human cardiomyopathy in which no aspects similar to those found in the Syrian hamster were observed (38). As far as electron microscopy is concerned, human and Syrian hamster cardiomyopathies therefore appear quite different. This latter disease, although genetically determined, is probably closer to ischemic heart disease than to other types of primary cardiomyopathies encountered in human patients.
Acknowledgements
We thank Dr. Jasmin for fruitful discussions and for supplying us with the animals used in this study. We acknowledge Ms. M. T. Dronne for excellent secretarial assistance. This work was supported in part by a grant from the A.M.F.
References
1. Adomian GE, Laks MM, Morady F, Swan HJC (1974) Significanceof the multiple intercalated disc in the hypertrophied canine heart. J Mol Cell Cardiol 6:105-110 2. Anversa P, Hagopian M, Loud AV (1973) Quantitative radioautographic localization of protein synthesis in experimental cardiac hypertrophy. Lab Invest 29:282-292 3. Bajusz E (1969) Hereditary cardiomyopathy: a new disease model. Am Heart J 77:686-696 4. Bajusz E, Baker JR, Nixon CW, Homburger F (1969) Spontaneous hereditary myocardial degeneration and congestive heart failure in a strain of Syrian hamsters. Ann N Y Acad Sci 156:105-129 5. Bishop SP, Cole CR (1969) Ultrastructural changes in canine myocardium with right ventricular hypertrophy and congestive heart failure. Lab Invest 20:219-229 6. B6hm M, Mende U, Schmitz W, Scholz H (1986) Increased responsiveness to stimulation of alpha but not beta-adrenoreceptors in the hereditary cardiomyopathy of the Syrian hamster. Intact adenosine and cholinoceptor mediated isoprenaline antagonist effect. Eur J Pharmacol 128:195-203 7. Breish EA (1984) Myocardial characteristics of pressure overload hypertrophy - a structural and functional study. Lab Invest 51:333-342 8. Brodsky WY, Uryvaeva IV (1977) Cell polyploidy: its relation to tissue growth and function. Int Rev Cytol 50:275-332
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63
9. Campbell SE, Gerdes AM, Smith TD (1987) Comparison of regional differences in cardiac myocyte dimensions in rats, hamsters, and guinea pigs. Anat Rec 219:53-59 10. Cluzeaud F, Perennec J, De Amoral E, Willemin M, Hatt PY (1984) Myocardial cell nucleus in heart overloading in the rat. Eur Heart J 5 (Suppl F):271-280 11. Csapo Z, Dusek J, Rona G (1972) Early alterations of the cardiac muscle cells in isoproterenolinduced necrosis. Arch Pathol 93:356-365 12. Factor SM, Sonnenblick EH (1985) The pathogenesis of clinical and experimental congestive cardiomyopathies: recent concepts. Prog Cardiovasc Dis 27:395-420 13. Ferrans VJ, Jones M, Maron B J, Roberts WC (1975) The nuclear membranes in hypertrophied human cardiac muscle celIs. Am J Pathol 78:427-460 14. Galle J, Mohr W, Lossnitzer K, Haferkamp O (1981) Development of necrosis and its sequelae in the myocardium of polymyopathic hamsters (Bio 8262). Virchows Arch (Cell Pathol) 36:87-100 15. Gerdes AM, Moore JA, Hines JM, Kirkland PA, Bishop SP (1986) Regional differences in myocyte size in normal rat heart. Anat Rec 215:420426 16. Hatt PY (1977) Cellular changes in mechanically overloaded heart. Basic Res Cardiol 72:158-202 17. Hatt PY, Jouannot P, Moravec J, Perennec J, Laplace M (1978) Development and reversal of pressure-induced cardiac hypertrophy. Light and electron microscopic study in the rat under temporary aortic constriction. Basic Res Cardiol 73:405-421 18. Hatt PY, Cluzeaud F, Perennec J (1982) Left ventricular hypertrophy - experimental aspects. Eur Heart J 3 (Suppl A):9-14 19. Jasmin G, Bajusz E (1973) Polymyopathie et cardiomyopathie h6r6ditaire chez le hamster de Syrie. Inhibition s61ective des 16sions du myocarde. Ann Anat Pathol 18(1):49-66 20. Karliner JS, Alabaster C, Stephens H, Barnes P, Dollery C (1981) Enhanced noradrenaline response in cardiomyopathic hamsters: possible relation to changes in adrenoceptors studied by radioligand binding. Cardiovasc Res 15:296-304 21. Korecky B, Sweet S, Rakusan K (1979) Number of nuclei in mammalian cardiac myocytes. Can J Physiol Pharmacol 57:1122-1129 22. Laks MM, Morady F, Adomian GE, Swan HJC (1970) Presence of widened and multiple intercalated discs in the hypertrophied canine heart. Circ Res 27:391-401 23. Lazarus ML, Colgan JA, Sachs HG (1976) Quantitative light and electron microscopic comparison of the normal and cardiomyopathic Syrian hamster heart. J Mol Cell Cardiol 8:431-441 24. Legato MJ (1970) Sarcomerogenesis in human myocardium. J Mol Cell Cardiol 1:425-437 25. Legato MJ (1984) The ultrastructure of myocardial hypertrophy - why does the compensated heart fail? Eur Heart J 5 (Suppl F):251-269 26. Leyton RA, Sonnenblick EH (1969) The ultrastructure of the failing heart. Am J Med Sci 258:304-327 27. Liew CC, Sole MJ (1978) Studies of nuclear proteins in the heart of the cardiomyopathic Syrian hamster - phosphorylation of histones. J Mol Cell Cardiol 10:847-855 28. Lossnitzer K, Janke J, Hein B, Stauch M, Fleckenstein A (1975) Disturbed myocardial metabolism a possible pathogenic factor in hereditary cardiomyopathy of the Syrian hamster. Recent Adv Stud Cardiac Struct Metab 6:283-290 29. Makino N, Jasmin G, Beamish RE, Dhala NS (1985) Sarcolemmal Na+-Ca + exchange during the development of genetically determined cardiomyopathy. Biochem Biophys Res Comm 133:491-497 30. Maron BJ, Ferrans VJ (1973) Significance of multiple intercalated discs in hypertrophied human myocardiuln. Am J Pathol 73:81-96 31. Maron B J, Ferrans VJ, Roberts WC (1975) Myocardial ultrastructure in patients with chronic aortic valve disease. Am J Cardiol 35:725-739 32. Mohr W, Lossnitzer K (1974) Morphologische Untersuchungen an Hamstern des Stammes BIO 8262 mit erblicher Myopathie und Kardiomyopathie. Beitr Pathol Bd 153:178-193 33. Nag AC, Cheng M (1987) Isolation long-term culture and ultrastructural characterization of adult cardiomyopathic cardiac muscle cells. In Vuurg Cell Develop Biol 23:261-266 34. Olbrich HG, Borgers M, Thone F, Frotscher M, Mutschler E, Schneider M, Kober G, Kaltenbach M (1988) Ultrastructural localization of calcium in the myocardium of cardiomyopathic Syrian hamsters. J Mol Cell Cardiol 20:753-762 -
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35. Onishi S, Btichner F, Zittel R (1969) Das elektronenmikroskopische Bild der Herzmuskelzelle des Hundes bei experimenteller Herzhypertrophie in der Anpassungsphase. Beitr Path Anat 139:94-114 36. Panagia V, Singh JN, Anand-Srivastava MB, Pierce GN, Jasmin G, Dhalla NS (1984) Sarcolemmal alterations during the development of genetically determined cardiomyopathy. Cardiovasc Res 18:567-572 37. Paterson RA, Layberry RA, Nadkarni BB (1972) Cardiac failure in the hamster. A biochemical and electron microscopic study. Lab Invest 26:755-766 38. Perennec J, Willemin M, Baraer P, Hart PY (1990) Comparative morphologic and morphometric study of the myocardium in human cardiomyopathy and aortic insufficiency. J Mol Cell Cardiol 22 (Suppl IV):S48 (abstract) 39. Poche R (1968) Ultrastructure of heart muscle under pathological condition. Ann N Y Acad Sci 156:1-625 40. Rona G (1985) Catecholamine cardiotoxicity. J Mol Cell Cardiol 17:291-306 41. Sole MF, Factor SM (1984) Hamster cardiomyopathy a genetically transmitted sympathetic dystrophy? In: Beamish RE, Panagia V, Dhalla NS (eds) Pathogenesis of Stress Induced Heart Disease. Martinus Nyhoff, Boston, pp 34M3 42. Sonnenblick EH, Spotnitz HM, Spiro D (1964) Role of the sarcomere in ventricular function and the mechanism of heart failure. Circ Res XIV and XV (suppl II):II-70-II-81 43. Sorenson AL, Tepper D, Sonnenblick EH, Robinson TF, Capasso JM (1985) Size and shape of enzymatically isolated ventricular myocytes from rats and cardiomyopathic hamsters. Cardiovasc Res 19:793-799 44. Venkatakrishnan R, Rourke AW (1979) The structure, function, and turnover of cardiac myosin in normal and myopathic Syrian hamsters. Lab Invest 41:198-205 45. Wagner JA, Reynolds IJ, Weisman HF, Dubeck P, Wersfeldt ML, Snyder SH (1986) Calcium antagonist receptors in cardiomyopathic hamsters: selective increases in heart muscle brain. Science 232:515-518 46. Weibel ER, Bolender RP (1974) Stereological techniques for electron microscopic morphometry. In: Hayat MA (ed) Principles of Electron Microscopy, Biological Application. Reinhold Company, Van Nostrand, pp 239-299 Received July 3, 1991 accepted December 2, 1991 Authors' address: J. Perennec, I.N.S.E.R.M. U2, H6pital L6on Bernard, 94456 Limeil-Brdvannes C6dex, France
Cardiology
Basic Res Cardio187:54-64
(1992)
Cardiac ultrastructural abnormalities in Syrian hamsters with spontaneous cardiomyopathy or subjected to cardiac overloads J. Perennec, M. Willemin, P. Pocholle, P. Y. Hatt, and B. Crozatier I.N.S.E.R.M. U2, H6pital Ldon Bernard, Limeil-Br6vannes, France Summary: Ultrastructural and morphometric abnormalities of Syrian hamster cardiomyopathy were compared to those observed in two different models of cardiac hypertrophy produced by mechanical overload (abdominal aortic stenosis, 60-day duration) or by isoproterenol injection during 15 days in normal Syrian hamsters of the same strain. Aspects of increased protein synthesis were observed in all three groups of animals. This was the only abnormality observed in the aortic stenosis group. Cardiomyopathy was different from the two other types of overload by the existence of large calcium deposits inside of the myocytes, by the presence of thin filaments and amorphous material accumulation suggesting abnormal synthesis and by a significant reduction of myofibrils at the heart-failure phase. Nuclear abnormalities with nuclear constrictions suggesting a division process and an increased number of myocytes with two nuclei were present in both spontaneous cardiomyopathy and isoproterenolinduced cardiopathy. Therefore, Syrian hamster cardiomyopathy appears to be different from cardiopathy induced by hemodynamic overload but, in spite of specific aspects, resembles that induced by isoproterenol injections, strengthening the hypothesis of a pathogenic role of catecholamines in the Syrian hamster cardiomyopathy. Key words: Syrian hamster; cardiomyopathy; nuclear abnormalities Introduction
Few reproducible models of cardiomyopathies are available in experimental animals. The most commonly utilized model is the cardiomyopathy of the Syrian hamster, which is a genetically determined disease with three stages of evolution: an ischemic phase followed by a compensated hypertrophy stage which terminates in congestive heart failure (4). A number of morphological studies of the Syrian hamster cardiomyopathy have already been published (3, 14, 19, 23, 34, 35, 37). However, no study determined which alterations were specific for cardiomyopathy and which alterations could be attributed to ischemia and to the hypertrophy process. The goal of our work was to analyze morphological alterations of the cardiomyopathic Syrian hamster in comparison with other cardiac alterations obtained in animals of the normal strain subjected to isoproterenol-indueed ischemia and to pressure-overload hypertrophy. The ultimate goal of our study was to attempt to determine the extent to which the genetically transmitted disease of the Syrian hamster simulated the observed forms of cardiopathies in patients (idiopathic cardiomyopathies or cardiac overloads due to hemodynamic alterations or to ischemic heart disease). Material and Method
1 Animals
Cardiomyopathic Syrian hamsters (UM-X 7.1 strain derived from Bio 14.6, Dr. Jasmin, Universit6 de Montr6al, Qu6bec, Canada) included 90-day-old animals (hypertrophy stage, n = 12) and 150-180707
Perennec et al., Syrian hamster cardiac ultrastructure
55
day-old animals (cardiac insufficiency stage, n = 14). They were compared to 20 healthy hamsters of the same age and of the same strain. An abdominal aortic constriction was performed in order to produce hypertrophy in another series of 13 healthy 45-day-old hamsters. The vessel was constricted above renal arteries with a plastic ring for producing a reduction of the aortic lumen by approximately 50-60 %. The animals were sacrificed 60 days after this constriction. Ten healthy animals of the same age served as controls for this series of animals. Cardiac hypertrophy was also produced in 15 healthy Syrian hamsters 60 days of age by daily subcutaneous injection of isoproterenol hydrochloride (0.5 mg/kg body weight) during 15 days. They were then sacrificed and compared to 10 controls of the same age. All animals (hypertrophied and cardiomyopathic) were sacrificed at the same age.
2 Fixation and tissue preparation Animals were anesthetized with intraperitoneal injection of pentothal (30 mg/kg of body weight). After cannulation of the abdominal aorta, heart arrest was induced in diastole by injection of procainamide in the IVC. Animals were retrogradedly perfused through the abdominal catheter with a fixation medium (1.25 % paraformaldehyde, 1.25 % glutaraldehyde buffered in cacodylate 50 mM with CaCla 4.1 raM; pH 7.4) for 5 rain under a perfusion pressure of 120 mmHg. After removal of the hearts from the thorax, transmural tissue samples were taken from the anterior, lateral, and posterior left-ventricular free walls. Tissue samples were immersed in the fixation medium, rinsed in cold sodium cacodylate buffer during 1 h, then postfixed in 2 % osmium tetroxyde for i h, and dehydrated in a graded ethanol series. Substitution of ethanol by propylene oxide was followed by incubation of the tissue in a mixture of propylene oxyde and epon. After 1 h the tissue was embedded in epon and polymerized for 24 h at 60 ~ Semi-thin sections were cut from each block, stained with blue toluidine and examined under a Zeiss microscope. Thin sections ( < 0.1 ~m) were prepared, mounted on uncovered copper grids, stained with uranyl acetate and lead citrate, and observed under a Zeiss EM 9 microscope.
3 Quantitative analysis Fiber diameter was determined using a micrometric ocular microscope. Longitudinal semi-thin sections were selected and examined at a magnification of x 500. Diameters were determined at the level of the nuclei; 250 measurements per sample were performed. Number of nuclei per cell was determined by examination of longitudinal semi-thin sections at a magnification of x 500; 250 myocytes per sample were examined. The fraction of cell volume occupied by mitochondria, myofibrils, and sarcoplasm was determined by the point-counting analysis described by Weibel (46), using a grid composed of intersecting lines 1-cm apart and placed upon an electron micrograph at a final magnification of • A minimum of 2000 points was counted per animaI. Volume fractions of subcellular components were calculated by dividing the number of points overlying a given component by the total points overlying the tissue. All data of different groups of animals were compared statistically using a one-way analysis of variance for multiple means. When a significant trend was found by the F test, Student's t-test was used to compare two different means with a correction by Bonferoni's method of multiple means. Results were considered for p < 0.05. Results
1 Morphological data W e o b s e r v e d lipid droplets inside myocytes in all groups, including the control group, w h e n animals were sacrificed in a u t u m n or in winter. Cardiomyopathic Syrian hamsters at the hypertrophy stage: No obvious myofibrillar alterations were seen in this group. F e a t u r e s evoking e n h a n c e d synthesis were c o m m o n : large nuclei with several nucleoli or p s e u d o vesicles, proliferative Golgi a p p a r a t u s , d e v e l o p e d sarcoplasmic reticulum, loci of m y o f i l a m e n t synthesis. A constriction of the middle p a r t of nuclei was a peculiar aspect o f t e n o b s e r v e d in this g r o u p of animals (Fig. 1).
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Fig. t. Electronmicrograph of nuclei of cardiomyopathic Syrian hamster at the hypertrophy stage, showing a peculiar aspect of the nucleus with an identation in its middle part. This aspect was often encountered (~ 2/~of cells). Mag.: x 10 000.
Cardiomyopathic Syrian hamsters at the cardiac insufficiency stage: In this group a number of alterations was observed in myofibrils, intercalated discs, mitochondria, T tubules and nuclei. Disorganization of myofibrillar arrangement was sometimes seen. Different degrees of structural alterations were observed. Z bands were often irregular with disappearance of their usual alignment in adjacent myofibrils. Z band material spread along the sarcomere in the direction of adjacent myofibrils (Fig. 2). During this end stage, features of synthesis and degeneration coexisted. Myofibrils were scarce in large myocytes. Foci of amorphous material or of thin filaments evoked abnormal synthesis (Fig. 3). Intercalated discs were very irregular, sometimes duplicated. We observed disappearance of thin filaments inserted upon them. This disappearance was predominant on one side of the intercalated disc, and T tubules proliferated. They were often filled with dense material. Mitochondrial size varied from very small to giant mitochondria. Abnormalities of nuclei described in the hypertrophic group were more frequent at the end stage of the disease. Amorphous circular concretions of calcium deposits occupied an important part of some cells adjacent to cells appearing normal (Fig. 4). Normal hamsters with aortic stenosis: Only features of synthesis were observed. Particularly large mitochondria were frequently noted. Nuclei were enlarged, but without indentation aspects. Normal hamsters o f the isoproterenolgroup: Foci of fibrosis were developed. Morphological aspect was similar to that of the stenosis group. Indentations of nuclei similar to those observed in the heart-failure group were sometimes, but rarely encountered.
Perennec et al., Syrian hamster cardiac ultrastructure
57
Fig. 2. Electronmicrograph of a cardiomyopathic Syrian hamster at the cardiac insufficiency stage. Z-band material spread in the direction of the membrane (arrow). Note calcium deposits (Ca) and collagen fibers (C). Mag.: x 22 000.
2
Quantitative data
Myofiber diameter was significantly larger in the four pathologic groups as compared with the control group (all Ps < 0.001; Table 1). In both the control group and the stenosis group the majority of myocytes had one nucleus. In contrast, in cardiomyopathic Syrian hamsters there were significantly more myocardial cells with two nuclei than cells with one nucleus. In the isoproterenol group, an intermediate result was found (Fig. 5). The morphometric study showed (Table 1) a significantly decreased myofibrillar volume in the cardiomyopathic hamsters during the ultimate stage only. It was related to the loss of myofibrils and mitochondrial volume being unchanged. In the other groups, results were similar to the controls. Discussion
Some of the presently described alterations were common to all types of pathological processes (genetically transmitted disease and cardiomyopathy induced by isoproterenol or pressure overload). In contrast, some alterations were specific for genetically derived disorder or were common with isoproterenol induced cardiopathy.
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Table 1. Morphometric data in Syrian hamsters.
Controls (n = 40) Cardiomyopathic (hypertrophy) (n = 12) Cardiomyopathic (cardiac insufficiency) (n = 14) Aortic stenosis (n = 13) Isoproterenol (n = 15) F
Myofiber diameters (~m)
V Myofibrils (%)
V Mitochondria
V Cytoplasm (%)
15.2 + 0.7
59.7 + 6.2
24.4 • 4.7
15.9 + 3.7
23.5 _+1.38*
57.1 _+7.8
23.9 + 5.6
18.9 _+4.8
23.4 _+ 1.7"
46.4 +_6.24
22.8 + 5.2
30.8 • 5.11
23.4 • 2.10
63.7 + 6.2
23.1 _+3.9
13.2 + 4.7
21.3 • 1.72"
57.7 • 5.1
23.1 _+3.8
19.2 • 4.3
p < 0.05
p < 0.001
NS
p < 0.001
Myofiber diameters and fractional volume occupied by myofibrils, mitochondria, and cytoplasm (mean • 1 s.d.) were compared in the five groups of Syrian hamsters (normal, cardiomyopathy at the compensated and heart failure stage and pressure and isoproterenol induced overload). *: p < 0.05; ~: p < 0.005 with the control group.
Cardiac alterations c o m m o n to all three types o f cardiopathies
The same degree of increase of cellular diameter was observed without modifications of the fractional volumes of mitochondria, myofibrils, and sarcoplasm in all three types of cardiopathies (Tables 1, 2) with features of increased protein synthesis similar to those described in mechanical overloads of other species (7, 15, 16). Larger values were found in control animals by Sorenson et al. (43). However, they used isolated cells which were not subjected to strains due to interstitial tissues. The increase in nucleus size is a commonly described pattern in a n u m b e r of clinical and experimental settings (13, 26, 39, 42). Similarly, a n u m b e r of alterations observed in myocardiopathic Syrian hamsters examined at the stage of heart failure were similar to those previousTy described in cardiopathies of other etiologies. Multiplication of intercalated discs was described in experimental and human cardiac overloads (1, 22, 25, 30) and had been attributed to the increased tension on cardiac cells due to the hemodynamic overload. Myofibrillar volume decrease is commonly observed in h u m a n heart failure due to hemodynamic overload (26). This pattern is in contrast with that described by Lazarus et al. (23) in the same experimental model as ours, but our results are in agreement with other descriptions of the hamster cardiomyopathy at the end-stage of heart failure (37). They can be compared to those of Venkatakrishnan et al. (44), who found an increased rate of myosin degradation with a loss of myosin. Degenerative aspects with myofibrillar lysis have also been described in h u m a n myocardium of patients with chronic aortic valve disease (31) and by Hart et al. (17) in rats subjected to pressure overload with aspects of disruption and clarification of intercalated discs. However, features of myofibrillar synthesis persisted in the cardiomyopathy of the Syrian hamster, even at this stage of heart failure. The association of aspects of synthesis and degeneration
Perennec et al., Syrian hamster cardiac ultrastructure
59
Fig. 3. Electronmicrograph of cardiomyopathic Syrian hamster at the cardiac insufficiency stage with loci of amorphous material (+) may be evoking abnormal synthesis or degraded material. This aspect was not encountered in controls. Mag.: • 10 000.
has been described in experimental cardiopathies induced by hemodynamic overload. Features evoking increased myofibrillar synthesis had been noted by Anversa et al. (2), Bishop et al. (5), Hatt et al. (18), and Legato (24) demonstrated an increased leucine incorporation in Z bands of rat hearts subjected to an aortic constriction. The spread of Zband material along the sarcomere (Fig. 4) is similar to that described by Hatt et al. (18) in rat pressure overload. It must be noted that all these experimental studies examined the myocardium at the compensated phase. In contrast with the persistence of features of myofibrillar synthesis in heart failure of the Syrian hamster, we (38) did not find such features in human cardiomyopathy studied at the end-stage of heart failure. In this respect, human cardiomyopathy is thus different from that of the Syrian hamster. Cardiac abnormalities specific to the inherited disease
A calcium accumulation in myocardial ceils was a specific feature of the cardiomyopathy of the Syrian hamster observed at the end-stage of heart failure (Figs. 4A and B). Although calcium accumulation has been described in myocardial cells in the early phase of Syrian hamster cardiomyopathy (32, 34) or in the connective tissue at the stage of heart failure (32), we did not find in the literature any description of intracellular calcium at this stage or large calcium concretions such as those found here (Figs. 4A and B). Intracellular calcium
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Fig. 4. Electronmicrograph of cardiomyopathic Syrian hamster at the cardiac insufficiency stage. A) calcium deposits (Ca) were observed adjacent to a myocyte appearing normal (My). Mag. x 5000. B) amorphous circular concretions of calcium (+). Mag.: x 20000.
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Perennec et al., Syrian hamster cardiac ultrastructure
overload has been considered as responsible for the initiation of myocardial lesions due to different reasons: sarcolemmal membrane permeability changes (29, 36, 45), increased sympathetic activity (6, 28, 29, 41) with an increased alpha-receptor number and response to norepinephrine (20), depression of the Na-K ATPase activity (29, 36), or an increased number of slow calcium channels (45). Cardiac abnormalities c o m m o n to inherited and isoproterenol-induced cardiomyopathies
Calcium accumulation was not observed in the isoproterenol-induced cardiopathy of the Syrian hamster, but a number of features were common to this type of cardiopathy and to the inherited disease. Thin filament accumulation suggesting a myofibrillar degradation was described both by Nag et al. (33) in cell cultures of cardiomyopathic Syrian hamster and in isoproterenol-induced ischemia in rats by Csapo et al. (11), who suggested a redistribution of actin. The number of cells with 2 nuclei was increased both in isoproterenol and inherited cardiopathy of the Syrian hamster as compared with normal hamsters in which 80 % of the cells had only one nucleus (Fig. 5). This species is thus different from other species in which cells with two nuclei predominate (21). This result is different from that of Campbell et al. (9) and of Nag et al. (33), who found a majority of ceils with two nuclei in cell cultures of normal Syrian hamsters. A possible explanation for this discrepancy is a selection of cells with two nuclei during cell isolation. Although such a selection has never been described, it is difficult to assess, but cannot be excluded. The most striking nuclear abnormality found in myocardiopathic Syrian hamsters was the aspect of nuclear indentation observed at the hypertrophy stage (Fig. 1), and even more frequently during the end stage of heart failure. It was never observed in the control group, and was not the result of a cellular contraction. The significance of this aspect is unknown. It can be explained by a cell fusion such as that shown
1OO
.[ L
Cells
with
1 nucleus
u z
-~
C e l l s w i t h 9, n u c l e i p < 0.01 with control
c~
g -r v-
50
g
CONTROL
A.S.
ISO.
H.C.M.
Fig. 5. Number of nuclei per cell. Bars indicate 1 sem. The percentage of cells with two nuclei was increased in the isoproterenol (ISO) and cardiomyopathy (HCM) groups as compared with the control group (* p < 0.01). The overall F value was highly significant (p < 0.001). A. S, = aortic stenosis group.
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Basic Research in Cardiology, Vol. 87, No. 1 (1992)
in young rats which leads to binuclear cells, to an acytokinetic mitosis similar to that found in the liver or to a polyteny, that is, a complete absence of mitotic mechanism leading to a duplication of DNA (8). This latter phenomenon would be in agreement with the findings of Paterson et al. (37) who found an increase of DNA which was larger than that of proteins without increases of endothelial or conjunctive cells in cardiomyopathic Syrian hamsters. Similarly, Liew et al. (27) found in the same disease an increased amount of histones, suggesting an increased gene activity. These nuclear aspects were also found in Syrian hamsters submitted to isoproterenol injections. A polyploidization was previously described in isoproterenoMnduced cardiopathies of rats (10, 11, 40). The similarity between nucleus abnormalities and thin filament accumulation found in cardiomyopathic Syrian hamsters and normal Syrian hamsters subjected to isoproterenol injections suggests that, as already proposed, the inherited disease of this species may be due to increased myocardial sympathetic activity with local elevation of norepinephrine concentration leading to loci of necrosis (12). In conclusion, it appears from our data that a number of electron microscopic aspects of the cardiomyopathy of the Syrian hamster (foci of amorphous material, nuclear abnormalities, calcium deposits) are different from those observed in hemodynamic overload of the same species. Nuclear abnormalities and thin-filament accumulation more resemble the aspects found in isoproterenoMnduced cardiomyopathy. The persistence of signs of protein synthesis in the Syrian hamster cardiomyopathy is different from its absence in human cardiomyopathy in which no aspects similar to those found in the Syrian hamster were observed (38). As far as electron microscopy is concerned, human and Syrian hamster cardiomyopathies therefore appear quite different. This latter disease, although genetically determined, is probably closer to ischemic heart disease than to other types of primary cardiomyopathies encountered in human patients.
Acknowledgements
We thank Dr. Jasmin for fruitful discussions and for supplying us with the animals used in this study. We acknowledge Ms. M. T. Dronne for excellent secretarial assistance. This work was supported in part by a grant from the A.M.F.
References
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