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Junctional complexes in regenerating endocardium
JOURNAL OF ULTRASTRUCTURE RESEARCH
79, 133-141 (1982)
Junctional Complexes in Regenerating Endocardium I HI~LI~NE TURCOTTE, MARC B A Z I N , AND M I C H E L B O U T E T 2
Department of Pathology, Laval University, School of Medicine, and Research Center, Laval Hospital, Ste-Foy, Quebec, Canada Received November 20, 1980, and in revised form November 16, 1981 Desmosome formation was unexpectedly observed in rat endocardium following the administration of isoproterenol hydrochloride (ISO). Endocardial alterations include rapid loss of endocardial cells and covering of ruptures with platelets, monocytes, polymorphonuclear leukocytes, and fibroblasts. Tight junctions were observed between endocardial cells and monocytes or fibroblast-like cells 8 hr after drug administration. Gap junctions and desmosomes were observed in regenerating rat endocardium after 24 hr. Desmosomes were observed between regenerating endocardial cells at different stages of maturation. This observation is of particular interest since desmosomes have not been described in normal rat endocardium. The mechanism(s) for desmosome formation after ISO administration has not been elucidated but their elaboration is associated with increased protein synthesis, development of numerous long and thin microvllli on the endocardial cell surface, and the accumulation of a granular material on the endocardial luminal surface.
The effects of ISO on myocardium and membrane permeability have been the subject of several morphological and biochemical studies (Boutet et al., 1976; Rona et al., 1959, 1977; Vorbeck et al., 1975; Wood et al., 1971). However morphological endocardial alterations following ISO administration have not been published. This communication describes endocardial modifications which occur after ISO administration. Particular interest has been paid to the formation of junctional complexes. We were able to observe desmosome formation in regenerating cells of rat endocardium following isoproterenol hydrochloride (ISO)induced cardiac necrosis. This observation is of particular interest since most studies carried out on desmosome formation were reported to occur in undifferentiated cells in the process of differentiation. MATERIALS AND METHODS
Materials. Forty-eight male Wistar rats weighing 250-300 g were used for these experiments: 32 isopro1 This study was supported by grants of the Medical Research Council of Canada, The Canadian Heart Foundation, and the J. C. Edwards Foundation. z Holder of a J. C. Edwards Foundation Professorship.
terenol-treated rats and 16 control rats. Isoproterenol hydrochloride (Winthrop) was injected subcutaneously (i.e., 45 mg/kg body wt) whereas control rats were injected with saline. Methods. Rats were sacrificed after light ether anesthesia 2, 4, 8, 12, 16, 24, 48, or 72 hr following treatment. Hearts were fixed by left ventricular perfusion with a mixture of paraformaldehyde and glutaraldehyde (Karnovsky, 1965). Samples for electron microscopic studies were selected from the midportion of the left ventricle, postfixed by immersion, washed in cacodylate buffer, osmicated, dehydrated in alcohol, and embedded in Epon (Hfittner et al. 1973). Heart tissue was embedded in fiat molds facilitating the orientation of endocardial cells. Half of the samples were treated " e n bloc" with uranyl acetate (Karnovsky, 1965). All sections were stained with lead citrate (Venable and Coggeshall, 1965) and examined with an AEI electron microscope. For light microscopy, routine studies were carried out after fixation in Bouin's solution. RESULTS
A . Control R a t E n d o c a r d i u m
Endocardium from control rats was morphologically similar to that described in the literature. Our observations pay particular reference to membrane specializations including junctional complexes, microvilli, and plasmalemmal vesicles. Light microscope examination of endo133 0022-5320/82/050133-09502.00/t) Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Light micrograph of control rat endocardium. Note the thin endocardial cells with few prominent nuclei (arrows). Toluidine blue. × 700. FI6.2. TEM micrograph of control rat endocardium with flat cells showing rare intracytoplasmic organelles and many plasmalemmal vesicles. Note the flat nucleus close to an intercellular junctional complex. × 13 000. FIc. 3. TEM micrograph of control rat endocardium. Note the tight junction (arrow) within the intercellular cleft. × 45 000. FtG. 4. TEM micrograph of conti'ol rat endocardium. Note the gap junction (arrow) within the intercellular cleft. × 78 000.
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cardium from control rats revealed a con- protruded into the lumen of the ventricular tinuous cell layer comprising flat cells in chamber. which the nucleus was more or less promLight microscope examination of animals inent depending on whether the heart was sacrificed 24, 48, and 72 hr after ISO adminfixed during contraction or relaxation (Fig. istration revealed an endocardium compris1). ing thickened cells and a prominent nucleus Transmission electron microscope ex- which frequently contained more than one amination of endocardium from Wistar rats nucleolus. Cellular modifications were revealed fiat cells which were limited by a more pronounced from 24 to 72 hr. These unit membrane. endocardial lesions were not always related Intracytoplasmic components, The en- to numerous necrotic foci observed in the docardial cell cytoplasm contained an elon- myocardium (Figs. 5 and 6). gated flat nucleus surrounded by few cyMembrane specialization in regenerated toplasmic organelles. The nucleus generally rat endocardium. Endocardial cell loss afcontained one nucleolus. Mitochondria ter 2 to 16 hr occurred near intercellular were not numerous; they were small and clefts. The remaining intercellular junctionhad few cristae. The Golgi apparatus and al complexes were normal (i.e., tight and rough endoplasmic reticulum (RER) were gap junctions). Ruptures in the endocardial poorly developed. Few free ribosomes, cell layer were patched with remaining enmultivesicular bodies, and coated vesicles docardial cells, monocytes, and fibroblast(Fig. 2) were observed. like cells. Transmission electron microscope exMembrane specializations. Intercellular membrane specializations comprised nu- amination of rat endocardium 24 hr after merous tight and few gap junctions (Figs. ISO administration revealed membrane 3 and 4). At the luminal surface of the en- junctional specializations between regendocardial cells few short and scattered mi- erated endocardial ceils which consisted of crovilli were observed with respect to the gap and tight junctions and numerous desnumber of nuclei and intercellular bound- mosomes. Newly formed d e s m o s o m e s aries. Finger-like cytoplasmic projections were observed at different stages of matuwere often observed overlying the intercel- ration (Figs. 7 to 12). Some areas of the lular cleft and neighboring endocardial cell. cytoplasm near the intercellular membrane The luminal and abluminal endocardial cell were condensed. These condensations surface showed numerous plasmalemmal were generally symmetrical in both cells; vesicles which opened into the lumen of the occasionally a single cell was involved endocardial cavity and subendocardial (Figs. 8 to 10). In addition to cytoplasmic condensation in numerous cells, filaments space. Endocardial cells were observed lying on were observed perpendicular to the intera basement membrane. The narrow sub- cellular cleft membranes. These filaments endocardial space consisted of granular were not necessarily symmetrically formed ground substance and several reticulocol- in both cells. Several cells presented more mature desmosomes and exhibited cytolagenic fibers. plasmic densities and filaments (Fig. 12). B. Isoproterenol-Treated Rat Endocardium Intercellular filamentous structures were Subendocardial edema was observed by also observed perpendicular to cytoplasmic light microscopic examination in rats sac- membranes. Some desmosomes were short rificed 2 to 16 hr after ISO administration. while others were more extended. DesmoEndocardial cells from animals sacrificed somes were present and in proximity to an after 8 to 12 hr were thickened and nuclei enlargement of the intercellular space.
136
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Fie. 5. Light micrograph of endocardial cell layer 24 hr after ISO administration which shows a number of thick cells (arrow) overlying a necrotic area. Note the normal appearance of myocardial cells in the center of the micrograph. Toluidine blue. x 700. FIG. 6. Light micrograph of isoproterenol-treated rat after 72 hr which shows a thick endocardial cell layer overlying a necrotic area. Note the presence of many nucleoli within endocardial cell nuclei. Toluidine blue. x 1100.
JUNCTIONAL COMPLEXES IN ENDOCARDIUM
They were abundant after 24 hr; however, after 48 hr numerous desmosomes were observed in the intercellular clefts of regenerated endocardial cells. After 72 hr most of the desmosomes exhibited characteristics of fully developed structures. It was of interest to note that most of the desmosomes were observed between thickened cells that showed signs of enhanced protein synthesis. Microvilli in ISO-treated rats were more numerous and longer than in control animals. After 72 hr microvilli were distributed throughout most of the endocardial luminal surface and were more abundant than at 24 and 48 hr. Microvilli were often concentrated near nuclei and intercellular boundaries. High-power examination of transversally sectioned microvilli revealed subunits which appeared as small osmiophilic dots distributed along the inner circumference of the membrane (Fig. 12). These subunits were equidistant. Longitudinal sections revealed the filamentous-like structure of the subunits. A granular material was observed covering regenerated endocardial cells and between microvilli; the granular material was more abundant 48 and 72 hr after ISO administration. An irregular clear space often separated the granular material from the endocardial cell surface. Numerous plasmalemmal vesicles were observed within thickened endocardial cells. Some vesicles openings of the luminal surface were filled with the granular material. I n t r a c y t o p l a s m i c c o m p o n e n t s . Thickened regenerated endocardial cells from ISO-treated animals showed signs of increased protein synthesis: the Golgi apparatus and RER were well developed. Dilatations in the RER were filled with a secretory-like material. Mitochondria were
137
abundant and showed an osmiophilic matrix with numerous cristae. Free ribosomes and glycogen granules were prominent (Figs. 7 to 12). Nuclei were enlarged and often contained more than one nucleolus. The subendocardial space was enlarged and comprised a more osmiophilic granular material than that observed on the endocardial luminal surface. DISCUSSION
Existing studies on the endocardium are few and describe normal endocardium in the rat, dog, rabbit, and frog (Anversa et al., 1975; Buss et al., 1973; Buss and Hollweg, 1977; Candiollo, 1963; Gavin et al., 1973; Mercer, 1965; Peine and Low, 1958; Sarphie and Allen, 1978; Shimamoto et al., 1969; Stehbens and Meyer, 1965; Wheeler et al., 1972). Our observations of normal rat endocardium were consistent with previously published reports (Anversa et al., 1975; Buss et al., 1973; Buss and Hollweg, 1977; Candiollo, 1963; Melax and Leeson, 1967). Junctional specializations in normal rat endocardium (i.e., tight and gap junctions) are similar to previous descriptions in vascular endothelium (Anversa et al., 1975; Buss et al., 1973; Buss and Hollweg, 1977; Candiollo, 1963; Gavin et al., 1973; Melax and Leeson, 1967; Muir and Peters, 1962; Peine and Low, 1958; Sarphie and Allen, 1978; Stehbens and Meyer, 1965; Wheeler et al., 1972; Giacomelli et al., 1975; Hfittner et al., 1973; Muir and Peters, 1962; Yee and Revel, 1975). To our knowledge endocardial modifications following isoproterenol administration have not previously been described. Regenerating endocardium was made by the interaction of endocardial cells monocytic and fibroblast-like cells. Changes ob-
FIG. 7. TEM micrograph of isoproterenol-treated rat after 48 hr which shows numerous microvilli in the intercellular cleft exhibiting many areas of membranous condensations. × 34 000. F~6. 8. TEM micrograph of 48-hr isoproterenol-treated rat. Note the thickened endocardial cells joined at the intercellular cleft by a short but mature desmosome. Note the widening of the intercellular space. × 24 800.
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TURCOTTE, BAZIN, AND BOUTET
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Fro. 9. TEM micrograph of 48-hr isoproterenol-treated rat. Note the asymmetrical desmosome forming within the intercellular cleft (arrow). Cytoplasmic filamentous structures are visible within the cell to the right. Inset: High-power magnification of the junctional complex. Fig. 9, × 36 000; inset, × 192 000. FIG. 10. TEM micrograph of 48-hr isoproterenol-treated rat. Note the asymmetrical desmosome between the two cells and the widening of the intercellular space behind the junctional complex. × 22 500. FIG. 11. TEM micrograph of 72-hr isoproterenol-treated rat. Note the hypertrophied Golgi apparatus (G),
JUNCTIONAL COMPLEXES IN ENDOCARDIUM
139
FIG. 12. High-power magnification of a fully developed desmosome with intermembranar perpendicular filaments and cytoplasmic filamentous condensations (ISO 48 hr). Note numerous microvilli sectioned transversally and longitudinally exhibiting filamentous internal structures and surrounded by a granular material. x 70 000.
served in regenerating endocardium also in- between regenerated endocardial cells were clude desmosome formation, increase in observed in ISO-treated rats sacrificed after the number of length of microvilli, and ul- 24, 48, and 72 hr. Fully developed desmotrastructural signs of increased protein syn- somes were similar to those previously dethesis. Desmosomes have been described scribed (Campbell and Campbell, 1971; in the capillary endothelium of rete mirabile Farquhar and Palade, 1963; McNutt, 1970; of fish swimbladder (Fawcett, 1961, 1963) McNutt and Weinstein, 1973). The rapid and in rainbow trout gill endothelium (Vo- formation of desmosomes in regenerated gel et al., 1976). Junctional specialization endocardium is in agreement with other of the desmosome type have not previously published studies (Buch and Krishan, 1965; been mentioned for mammalian endocar- Heaysman and Pegrum, 1973; Lloyd et al., 1976; Reinhardt et al., 1976). Reinhardt et dium or vascular endothelium. Various stages of desmosome formation al. (1976) demonstrated that desmosomes
numerous profiles of rough endoplasmic reticulum, and abundant mitochondria. There are numerous microviUi surrounded by a granular material at the luminal surface of the intercellular cleft. Note also the short desmosome (arrow) between two endocardial cells. Inset: High-power magnification of the junctional complex. Fig. 11, × 12000; inset, × 46000.
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were formed in regenerated drosophila wing imaginal discs immediately after cell contact was established. D e s m o s o m e s were easily identified during fibroblast aggregation after less than a minute (Heaysman and Pegrum, 1973; Lloyd et al., 1976). Desmosomes observed after ISO administration were more or less extended and generally symmetrical, h o w e v e r some showed varying degrees of asymmetry. The different desmosomal aspects correspond with varying stages in the formation of junctional specializations described by other investigators (Anversa et al., 1975; Deane and Wurzelmann 1965; Kelly, 1966; Mercer, 1965; Overton, 1962; Overton and Shoup, 1964; Patrizi, 1967a,b; Pease, 1966; Pflugfelder and Schubert, 1965; Rambourg and Leblond, 1967; Sternlieb, 1968; Trelstad et al., 1967; Wartiovaara, 1966; Weiss, 1957; Wood, 1965). Campbell and Campbell (1971) suggested that the activity of adjacent cells was synchronized so that desmosomes were produced symmetrically unless they were formed between different cell types. In ISO-treated rats desmosome symmetry was not a rule; it seemed that factors responsible for synchronization of desmosome formation were only partially active. Desmosomes were also produced between cells of various origins (i.e., fibroblast-like cell or regenerated endocardial cell and a remaining endocardial cell). It may be possible that endocardial cells, which are fully differentiated cells, are unable to control the synchronization of desmosome elaboration. However it may also be due to the tangential orientation of the tissue. Two theories regarding desmosome formation have been suggested. First, desmosomes may be produced by the cellular synthesis of their constituents. Second, specific membrane regions have chemical and physical properties which permit the transformation of membranous material into desmosome components (Campbell and Campbell, 1971; Mercer, 1965). Desmosome in regenerated endocardial cells
from ISO-treated rats may be synthesized from cellular materials since many cells showed an increased number of mitochondria, free ribosomes, well-developed Golgi apparatus, and RER. The granular material observed on the luminal surface of the endocardium may have been synthesized by regenerated endocardial cells. While many investigations exclude the hypothesis of cellular synthesis of desmosome components our results support this theory; however, we were unable to provide adequate explanation for the synthesis of elements which constitute the desmosome. Besides, ISO has been known to stimulate DNA synthesis (Barka, 1965; Baserga and Heftier, 1967; Vorbeck et al., 1975; Wood et al., 1971). Initiation of desmosome production in ISO-treated regenerated endocardium may be a result of the direct or indirect action of isoproterenol or its oxidation products on protein synthesis. In summary injection of ISO into Wistar rats promotes the formation of tight and gap junctions and desmosomes between regenerating endocardial cells. Factors responsible for the formation of desmosome constituents and their subsequent organization into desmosomes in endocardial cells of various tissues remain unknown. REFERENCES ANVERSA, P., G1ACOMELLI, F., AND WIENER, J. (1975) Anat. Res. 183, 477--481. BARKA, T. (1965) Exp. Cell Res. 39, 355-364. BASERrA, R., AND HEFFLER, S. (1967) Exp. Cell Res. 46, 571-580. BOUX~T, M., HOTTNER,I., ANORONA, G. (1976) Lab. Invest. 34, 382-388. BucH, R. C., AND KR~SIJAN,A. (1965) Exp. Cell Res. 38, 426-428. BUSS, H., DAHM,H. H., AND LIDENFELSER,R. (1973) Beitr. Pathol. 148, 340-359. Buss, H., AND HOLLWE6, H. G. (1977) Scanning Electron Microsc. 2, 467-476. CAMPBELL, R. D., ANO CAMeBELL,J. H. (1971) Results and Problems in Cell Differentiation: Origin and Continuity of Cell Organelles, pp. 261-298, Springer-Verlag, New York. CANDIOLLO, L. (1963) Z. Zellforsch. 61,486--492. DEANE, H. W., AND WURZELMANN,S. (1965) J. Cell Biol. 27, 131a.
JUNCTIONAL COMPLEXES IN ENDOCARDIUM FARQUHAR, M. G., AND PALADE, G, E. (1963) J. Cell Biol. 17, 375-412. FAWCETT, D. W. (1961) Exp. Cell Res. Suppl. 8, 174-187. FAWCETT, D. W. (1963) The Peripheral Blood Vessels, pp. 17-44, Williams & Wilkins, Baltimore. GAVIN, J. B., WHEELER, E. E., AND HERDSON, P. B. (1973) Pathology 5, 145-148. GIACOMELLI, F., ANVERSA, P., AND WIENER, J. (1975) Microvasc. Res. 10, 38-42. HEAYSMAN,J. E. M., AND PEGRUM°S. M. (1973) Exp. Cell Res. 78, 71-78. HUTTNER, I., BOUTET, M., AND MORE, R. H. (1973) J. Cell Biol. 57, 247-252. KARNOVSKY, M. J. (1965)J. Cell Biol. 27, 137a. KELLY, D. E. (1966) J. Cell Biol. 28, 51-72. LLOYD, C. W., REES, D. A., SMITH, C. G., AND JUDGE, F. J. (1976) J. Cell Sci. 22, 671-684. McNUTT, N. S. (1970) Amer. J. Cardiol. 25, 169-183. McNUTT, N. S., AND WEINSTEIN, R. S. (1973) Prog. Biophys. Mol. Biol. 26, 45-101. MELAX, H., AND LEESON, T. S. (1967) Cardiovasc. Res. 1, 349-355. MERCER, E. H, (1965) Organogenesis, pp. 29-53, Holt, Rinehart & Winston, New York. MUIR, A. R., AND PETERS, A. (1962) J. Cell Biol. 12, 443-448. OVERTON, J. (1962) Develop. Biol. 4, 532-548. OVERTON, J., AND SHOUP, J. (1964) J. Cell Biol. 21, 75-85. PATRIZI, G. (1967a) Sperimentale 117, 189-203. PATRIZI, G. (1967b) J. Cell Biol. 35, 182a. PEASE, D. C. (1966) J. Ultrastruct. Res. 15, 555-588. PEINE, C. J., AND Low, F. N. (1958) Amer. J. Anat. 142, 137-158. PFLUGFELDER, O., AND SCHUBERT,G. (1965) Z. Zellforsch. 67, 96-112.
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RAMBOURG, A., AND LEBLOND, C. P. (1967) J. Cell Biol. 32, 27-53. REINHARDT, C. A., BRYANT, P. J., AND SCHEIDERMAN, H. A. (1976) J. Cell Biol. 70, 412a. RONA, G., CHAPPEL, C. I., BALAZS, T., AND GAUDav, T. (1959) Arch Pathol. 67, 443-455. RONA, G., HUTTNER, I., AND BOUTET, M. (1977) Handbuch der allgemeinen pathologie III/7 Microzirkulation/Microcirculation, pp. 791-888, SpringerVerlag, Berlin/Heidelberg/New York. SARPHIE, T. G., AND ALLEN, D. J. (1978) J. Submicrosc. Cytol. 10, 15-25. SHIMAMOTO, T., YAMASHITO, Y., AND SUNAGA, T. (1969) Proc. Jap. Acad. 45, 507-511. STEHBENS, W. E., AND MEYER, E. (1965) J. Anat. (London) 99, 127-135. STERNLIEB, I. (1968) Z. Zellforsch. 93, 249-253. TRELSTAD, R. L., HAY, E. D., AND REVEL, J. P. (1967) Develop. Biol. 16, 78-106. VENABLE, J. H., AND COGGESHALL,R. (1965) J. Cell Biol. 25, 407--408. VOGEL, W., VOGEL, V., AND PFAUTSCH, M. (1976) Cell Tissue Res. 167, 373-385. VORBECK, M. L., MALEWSKI, E. F., ERHART, L. S., AND MARTIN, A. P. (1975) in Recent Advances in Studies on Cardiac Structure and Metabolism, Vol. 6, pp. 175-181, University Park Pre~s, Baltimore. WARTIOVAARA, J. (1966) Ann. Med. Exp. Biol. Fenn. 44, 469-503. WEISS, P. (1957) Int. Rev. Cytol. 7, 391-423. WHEELER, E. E., GAVIN, J. B., AND HERDSON, P. B. (1972) Anat. Res. 175, 579-584. WOOD, R. L. (1965) Anat. Rec, 151,507-529. WOOD, W. G., L1NDENMAYER,G. E., AND SCHWARTZ, A. (1971) J. Mol. Cell Cardiol. 3, 127-139. YEE, A. G., AND REVEL, J. P. (1975) J. Cell Biol. 66, 200-204.
79, 133-141 (1982)
Junctional Complexes in Regenerating Endocardium I HI~LI~NE TURCOTTE, MARC B A Z I N , AND M I C H E L B O U T E T 2
Department of Pathology, Laval University, School of Medicine, and Research Center, Laval Hospital, Ste-Foy, Quebec, Canada Received November 20, 1980, and in revised form November 16, 1981 Desmosome formation was unexpectedly observed in rat endocardium following the administration of isoproterenol hydrochloride (ISO). Endocardial alterations include rapid loss of endocardial cells and covering of ruptures with platelets, monocytes, polymorphonuclear leukocytes, and fibroblasts. Tight junctions were observed between endocardial cells and monocytes or fibroblast-like cells 8 hr after drug administration. Gap junctions and desmosomes were observed in regenerating rat endocardium after 24 hr. Desmosomes were observed between regenerating endocardial cells at different stages of maturation. This observation is of particular interest since desmosomes have not been described in normal rat endocardium. The mechanism(s) for desmosome formation after ISO administration has not been elucidated but their elaboration is associated with increased protein synthesis, development of numerous long and thin microvllli on the endocardial cell surface, and the accumulation of a granular material on the endocardial luminal surface.
The effects of ISO on myocardium and membrane permeability have been the subject of several morphological and biochemical studies (Boutet et al., 1976; Rona et al., 1959, 1977; Vorbeck et al., 1975; Wood et al., 1971). However morphological endocardial alterations following ISO administration have not been published. This communication describes endocardial modifications which occur after ISO administration. Particular interest has been paid to the formation of junctional complexes. We were able to observe desmosome formation in regenerating cells of rat endocardium following isoproterenol hydrochloride (ISO)induced cardiac necrosis. This observation is of particular interest since most studies carried out on desmosome formation were reported to occur in undifferentiated cells in the process of differentiation. MATERIALS AND METHODS
Materials. Forty-eight male Wistar rats weighing 250-300 g were used for these experiments: 32 isopro1 This study was supported by grants of the Medical Research Council of Canada, The Canadian Heart Foundation, and the J. C. Edwards Foundation. z Holder of a J. C. Edwards Foundation Professorship.
terenol-treated rats and 16 control rats. Isoproterenol hydrochloride (Winthrop) was injected subcutaneously (i.e., 45 mg/kg body wt) whereas control rats were injected with saline. Methods. Rats were sacrificed after light ether anesthesia 2, 4, 8, 12, 16, 24, 48, or 72 hr following treatment. Hearts were fixed by left ventricular perfusion with a mixture of paraformaldehyde and glutaraldehyde (Karnovsky, 1965). Samples for electron microscopic studies were selected from the midportion of the left ventricle, postfixed by immersion, washed in cacodylate buffer, osmicated, dehydrated in alcohol, and embedded in Epon (Hfittner et al. 1973). Heart tissue was embedded in fiat molds facilitating the orientation of endocardial cells. Half of the samples were treated " e n bloc" with uranyl acetate (Karnovsky, 1965). All sections were stained with lead citrate (Venable and Coggeshall, 1965) and examined with an AEI electron microscope. For light microscopy, routine studies were carried out after fixation in Bouin's solution. RESULTS
A . Control R a t E n d o c a r d i u m
Endocardium from control rats was morphologically similar to that described in the literature. Our observations pay particular reference to membrane specializations including junctional complexes, microvilli, and plasmalemmal vesicles. Light microscope examination of endo133 0022-5320/82/050133-09502.00/t) Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Light micrograph of control rat endocardium. Note the thin endocardial cells with few prominent nuclei (arrows). Toluidine blue. × 700. FI6.2. TEM micrograph of control rat endocardium with flat cells showing rare intracytoplasmic organelles and many plasmalemmal vesicles. Note the flat nucleus close to an intercellular junctional complex. × 13 000. FIc. 3. TEM micrograph of control rat endocardium. Note the tight junction (arrow) within the intercellular cleft. × 45 000. FtG. 4. TEM micrograph of conti'ol rat endocardium. Note the gap junction (arrow) within the intercellular cleft. × 78 000.
J U N C T I O N A L C O M P L E X E S IN E N D O C A R D I U M
135
cardium from control rats revealed a con- protruded into the lumen of the ventricular tinuous cell layer comprising flat cells in chamber. which the nucleus was more or less promLight microscope examination of animals inent depending on whether the heart was sacrificed 24, 48, and 72 hr after ISO adminfixed during contraction or relaxation (Fig. istration revealed an endocardium compris1). ing thickened cells and a prominent nucleus Transmission electron microscope ex- which frequently contained more than one amination of endocardium from Wistar rats nucleolus. Cellular modifications were revealed fiat cells which were limited by a more pronounced from 24 to 72 hr. These unit membrane. endocardial lesions were not always related Intracytoplasmic components, The en- to numerous necrotic foci observed in the docardial cell cytoplasm contained an elon- myocardium (Figs. 5 and 6). gated flat nucleus surrounded by few cyMembrane specialization in regenerated toplasmic organelles. The nucleus generally rat endocardium. Endocardial cell loss afcontained one nucleolus. Mitochondria ter 2 to 16 hr occurred near intercellular were not numerous; they were small and clefts. The remaining intercellular junctionhad few cristae. The Golgi apparatus and al complexes were normal (i.e., tight and rough endoplasmic reticulum (RER) were gap junctions). Ruptures in the endocardial poorly developed. Few free ribosomes, cell layer were patched with remaining enmultivesicular bodies, and coated vesicles docardial cells, monocytes, and fibroblast(Fig. 2) were observed. like cells. Transmission electron microscope exMembrane specializations. Intercellular membrane specializations comprised nu- amination of rat endocardium 24 hr after merous tight and few gap junctions (Figs. ISO administration revealed membrane 3 and 4). At the luminal surface of the en- junctional specializations between regendocardial cells few short and scattered mi- erated endocardial ceils which consisted of crovilli were observed with respect to the gap and tight junctions and numerous desnumber of nuclei and intercellular bound- mosomes. Newly formed d e s m o s o m e s aries. Finger-like cytoplasmic projections were observed at different stages of matuwere often observed overlying the intercel- ration (Figs. 7 to 12). Some areas of the lular cleft and neighboring endocardial cell. cytoplasm near the intercellular membrane The luminal and abluminal endocardial cell were condensed. These condensations surface showed numerous plasmalemmal were generally symmetrical in both cells; vesicles which opened into the lumen of the occasionally a single cell was involved endocardial cavity and subendocardial (Figs. 8 to 10). In addition to cytoplasmic condensation in numerous cells, filaments space. Endocardial cells were observed lying on were observed perpendicular to the intera basement membrane. The narrow sub- cellular cleft membranes. These filaments endocardial space consisted of granular were not necessarily symmetrically formed ground substance and several reticulocol- in both cells. Several cells presented more mature desmosomes and exhibited cytolagenic fibers. plasmic densities and filaments (Fig. 12). B. Isoproterenol-Treated Rat Endocardium Intercellular filamentous structures were Subendocardial edema was observed by also observed perpendicular to cytoplasmic light microscopic examination in rats sac- membranes. Some desmosomes were short rificed 2 to 16 hr after ISO administration. while others were more extended. DesmoEndocardial cells from animals sacrificed somes were present and in proximity to an after 8 to 12 hr were thickened and nuclei enlargement of the intercellular space.
136
TURCOTTE, BAZIN, AND BOUTET d
• , . ~ . . e ~
5:
~"
:
Fie. 5. Light micrograph of endocardial cell layer 24 hr after ISO administration which shows a number of thick cells (arrow) overlying a necrotic area. Note the normal appearance of myocardial cells in the center of the micrograph. Toluidine blue. x 700. FIG. 6. Light micrograph of isoproterenol-treated rat after 72 hr which shows a thick endocardial cell layer overlying a necrotic area. Note the presence of many nucleoli within endocardial cell nuclei. Toluidine blue. x 1100.
JUNCTIONAL COMPLEXES IN ENDOCARDIUM
They were abundant after 24 hr; however, after 48 hr numerous desmosomes were observed in the intercellular clefts of regenerated endocardial cells. After 72 hr most of the desmosomes exhibited characteristics of fully developed structures. It was of interest to note that most of the desmosomes were observed between thickened cells that showed signs of enhanced protein synthesis. Microvilli in ISO-treated rats were more numerous and longer than in control animals. After 72 hr microvilli were distributed throughout most of the endocardial luminal surface and were more abundant than at 24 and 48 hr. Microvilli were often concentrated near nuclei and intercellular boundaries. High-power examination of transversally sectioned microvilli revealed subunits which appeared as small osmiophilic dots distributed along the inner circumference of the membrane (Fig. 12). These subunits were equidistant. Longitudinal sections revealed the filamentous-like structure of the subunits. A granular material was observed covering regenerated endocardial cells and between microvilli; the granular material was more abundant 48 and 72 hr after ISO administration. An irregular clear space often separated the granular material from the endocardial cell surface. Numerous plasmalemmal vesicles were observed within thickened endocardial cells. Some vesicles openings of the luminal surface were filled with the granular material. I n t r a c y t o p l a s m i c c o m p o n e n t s . Thickened regenerated endocardial cells from ISO-treated animals showed signs of increased protein synthesis: the Golgi apparatus and RER were well developed. Dilatations in the RER were filled with a secretory-like material. Mitochondria were
137
abundant and showed an osmiophilic matrix with numerous cristae. Free ribosomes and glycogen granules were prominent (Figs. 7 to 12). Nuclei were enlarged and often contained more than one nucleolus. The subendocardial space was enlarged and comprised a more osmiophilic granular material than that observed on the endocardial luminal surface. DISCUSSION
Existing studies on the endocardium are few and describe normal endocardium in the rat, dog, rabbit, and frog (Anversa et al., 1975; Buss et al., 1973; Buss and Hollweg, 1977; Candiollo, 1963; Gavin et al., 1973; Mercer, 1965; Peine and Low, 1958; Sarphie and Allen, 1978; Shimamoto et al., 1969; Stehbens and Meyer, 1965; Wheeler et al., 1972). Our observations of normal rat endocardium were consistent with previously published reports (Anversa et al., 1975; Buss et al., 1973; Buss and Hollweg, 1977; Candiollo, 1963; Melax and Leeson, 1967). Junctional specializations in normal rat endocardium (i.e., tight and gap junctions) are similar to previous descriptions in vascular endothelium (Anversa et al., 1975; Buss et al., 1973; Buss and Hollweg, 1977; Candiollo, 1963; Gavin et al., 1973; Melax and Leeson, 1967; Muir and Peters, 1962; Peine and Low, 1958; Sarphie and Allen, 1978; Stehbens and Meyer, 1965; Wheeler et al., 1972; Giacomelli et al., 1975; Hfittner et al., 1973; Muir and Peters, 1962; Yee and Revel, 1975). To our knowledge endocardial modifications following isoproterenol administration have not previously been described. Regenerating endocardium was made by the interaction of endocardial cells monocytic and fibroblast-like cells. Changes ob-
FIG. 7. TEM micrograph of isoproterenol-treated rat after 48 hr which shows numerous microvilli in the intercellular cleft exhibiting many areas of membranous condensations. × 34 000. F~6. 8. TEM micrograph of 48-hr isoproterenol-treated rat. Note the thickened endocardial cells joined at the intercellular cleft by a short but mature desmosome. Note the widening of the intercellular space. × 24 800.
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®
#
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Fro. 9. TEM micrograph of 48-hr isoproterenol-treated rat. Note the asymmetrical desmosome forming within the intercellular cleft (arrow). Cytoplasmic filamentous structures are visible within the cell to the right. Inset: High-power magnification of the junctional complex. Fig. 9, × 36 000; inset, × 192 000. FIG. 10. TEM micrograph of 48-hr isoproterenol-treated rat. Note the asymmetrical desmosome between the two cells and the widening of the intercellular space behind the junctional complex. × 22 500. FIG. 11. TEM micrograph of 72-hr isoproterenol-treated rat. Note the hypertrophied Golgi apparatus (G),
JUNCTIONAL COMPLEXES IN ENDOCARDIUM
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FIG. 12. High-power magnification of a fully developed desmosome with intermembranar perpendicular filaments and cytoplasmic filamentous condensations (ISO 48 hr). Note numerous microvilli sectioned transversally and longitudinally exhibiting filamentous internal structures and surrounded by a granular material. x 70 000.
served in regenerating endocardium also in- between regenerated endocardial cells were clude desmosome formation, increase in observed in ISO-treated rats sacrificed after the number of length of microvilli, and ul- 24, 48, and 72 hr. Fully developed desmotrastructural signs of increased protein syn- somes were similar to those previously dethesis. Desmosomes have been described scribed (Campbell and Campbell, 1971; in the capillary endothelium of rete mirabile Farquhar and Palade, 1963; McNutt, 1970; of fish swimbladder (Fawcett, 1961, 1963) McNutt and Weinstein, 1973). The rapid and in rainbow trout gill endothelium (Vo- formation of desmosomes in regenerated gel et al., 1976). Junctional specialization endocardium is in agreement with other of the desmosome type have not previously published studies (Buch and Krishan, 1965; been mentioned for mammalian endocar- Heaysman and Pegrum, 1973; Lloyd et al., 1976; Reinhardt et al., 1976). Reinhardt et dium or vascular endothelium. Various stages of desmosome formation al. (1976) demonstrated that desmosomes
numerous profiles of rough endoplasmic reticulum, and abundant mitochondria. There are numerous microviUi surrounded by a granular material at the luminal surface of the intercellular cleft. Note also the short desmosome (arrow) between two endocardial cells. Inset: High-power magnification of the junctional complex. Fig. 11, × 12000; inset, × 46000.
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were formed in regenerated drosophila wing imaginal discs immediately after cell contact was established. D e s m o s o m e s were easily identified during fibroblast aggregation after less than a minute (Heaysman and Pegrum, 1973; Lloyd et al., 1976). Desmosomes observed after ISO administration were more or less extended and generally symmetrical, h o w e v e r some showed varying degrees of asymmetry. The different desmosomal aspects correspond with varying stages in the formation of junctional specializations described by other investigators (Anversa et al., 1975; Deane and Wurzelmann 1965; Kelly, 1966; Mercer, 1965; Overton, 1962; Overton and Shoup, 1964; Patrizi, 1967a,b; Pease, 1966; Pflugfelder and Schubert, 1965; Rambourg and Leblond, 1967; Sternlieb, 1968; Trelstad et al., 1967; Wartiovaara, 1966; Weiss, 1957; Wood, 1965). Campbell and Campbell (1971) suggested that the activity of adjacent cells was synchronized so that desmosomes were produced symmetrically unless they were formed between different cell types. In ISO-treated rats desmosome symmetry was not a rule; it seemed that factors responsible for synchronization of desmosome formation were only partially active. Desmosomes were also produced between cells of various origins (i.e., fibroblast-like cell or regenerated endocardial cell and a remaining endocardial cell). It may be possible that endocardial cells, which are fully differentiated cells, are unable to control the synchronization of desmosome elaboration. However it may also be due to the tangential orientation of the tissue. Two theories regarding desmosome formation have been suggested. First, desmosomes may be produced by the cellular synthesis of their constituents. Second, specific membrane regions have chemical and physical properties which permit the transformation of membranous material into desmosome components (Campbell and Campbell, 1971; Mercer, 1965). Desmosome in regenerated endocardial cells
from ISO-treated rats may be synthesized from cellular materials since many cells showed an increased number of mitochondria, free ribosomes, well-developed Golgi apparatus, and RER. The granular material observed on the luminal surface of the endocardium may have been synthesized by regenerated endocardial cells. While many investigations exclude the hypothesis of cellular synthesis of desmosome components our results support this theory; however, we were unable to provide adequate explanation for the synthesis of elements which constitute the desmosome. Besides, ISO has been known to stimulate DNA synthesis (Barka, 1965; Baserga and Heftier, 1967; Vorbeck et al., 1975; Wood et al., 1971). Initiation of desmosome production in ISO-treated regenerated endocardium may be a result of the direct or indirect action of isoproterenol or its oxidation products on protein synthesis. In summary injection of ISO into Wistar rats promotes the formation of tight and gap junctions and desmosomes between regenerating endocardial cells. Factors responsible for the formation of desmosome constituents and their subsequent organization into desmosomes in endocardial cells of various tissues remain unknown. REFERENCES ANVERSA, P., G1ACOMELLI, F., AND WIENER, J. (1975) Anat. Res. 183, 477--481. BARKA, T. (1965) Exp. Cell Res. 39, 355-364. BASERrA, R., AND HEFFLER, S. (1967) Exp. Cell Res. 46, 571-580. BOUX~T, M., HOTTNER,I., ANORONA, G. (1976) Lab. Invest. 34, 382-388. BucH, R. C., AND KR~SIJAN,A. (1965) Exp. Cell Res. 38, 426-428. BUSS, H., DAHM,H. H., AND LIDENFELSER,R. (1973) Beitr. Pathol. 148, 340-359. Buss, H., AND HOLLWE6, H. G. (1977) Scanning Electron Microsc. 2, 467-476. CAMPBELL, R. D., ANO CAMeBELL,J. H. (1971) Results and Problems in Cell Differentiation: Origin and Continuity of Cell Organelles, pp. 261-298, Springer-Verlag, New York. CANDIOLLO, L. (1963) Z. Zellforsch. 61,486--492. DEANE, H. W., AND WURZELMANN,S. (1965) J. Cell Biol. 27, 131a.
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