Focal Laminate Segments in Cytoplasmic Processes of Mouse Myocardial Fibroblasts M. S. F O R B E S AND N. S P E R E L A K I S Department of Physiology, University of Virgznia School of Medrcine, Charlottesudle, Virginia 22908

ABSTRACT In mouse ventricular myocardium, we have found unusual fibroblasts whose cellular processes in some regions are particularly flattened and which contain linearly-arranged, electron-opaque structures (“central laminae”). The morphology of these focal laminate segments of fibroblast processes suggests that the intracellular laminae are adhesive entities which hold the plasmalemmata above and below them in close parallel apposition for short distances, A complex system of interconnected extracellular compartments is present throughout the mammalian heart. This system has been termed the “tissue space” (Melax and Leeson, ’72) and comprises the perivascular spaces, the subepicardial and subendocardial spaces, and the interstitial spaces between the cardiac muscle cells themselves. Within the myocardial tissue space, the most populous cells are fibroblasts (Melax and Leeson, ’72). Aside from their constituting a nuisance in the cuIture of heart muscle cells, myocardial fibroblasts have enjoyed little attention. However, we have found in mouse heart a unique structure, the “central lamina,” that occurs within particularly thin processes of fibroblasts. The fibroblastic segments containing such laminae are somewhat similar in configuration to flattened regions seen in various cell types in vitro (Conley and Herman, ’73; Franke et al., ’78). We conclude that such complexes represent sites of intracellular adhesion. MATERIALS A N D METHODS

The animals investigated were mice of either the ICR or C57BL strains. Each animal was anesthetized with an intraperitoneal injection of pentobarbital and its thoracic cavit y was opened, exposing the beating heart. Fixative solution was injected into the circulatory system through a butterfly infusion needle inserted into the apex of the left ventricle, and left the system via an incision in the right atrium. Fixative solutions used included: (1) 3% glutaraldehyde in aqueous 3% ANAT. REC. (1979) 195: 575-586.

dextrose-3% dextran, with or without 50 mM CaC1, added (modified from Rostgaard and Behnke, ’65); (2) 2%paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate with 50 mM CaC12 (McNutt and Weinstein, ’70); or (3) 2% paraformaldehyde and 3% glutaraldehyde in Hank’s balanced salt solution (Bois, ’73). After 5 minutes of perfusion fixation, the heart was removed, and portions were fixed an additional 3 hours by immersion in fixative solution. All tissues were postfixed in 1%phosphate-buffered osmium tetroxide (Millonig, ’621, stained en bloc for 30 minutes in saturated aqueous uranyl acetate, dehydrated in alcohols, passed through propylene oxide and embedded in Epon 812 resin. Thin sections were cut with a diamond knife, affixed to copper mesh grids and stained sequentially with saturated uranyl acetate in 50% acetone (2 min) and 0.4%alkaline lead citrate (45 sec) (Venable and Coggeshall, ’65). The sections were examined in either Philips EM200 or Zeiss EM 9A electron microscopes, which instruments were calibrated periodically against a replica of a diffraction grating. For critical measurements of structures, micrographs were taken a t high magnification, and the microscopes then calibrated at that magnification; photographs were prepared a t 10 x enlargement (approximately 400,000 X total magnification), and t h e cytological structures of interest were measured with vernier calipers. Some sections were analyzed by tilting in a Siemens 101 electron microscope equipped with a goniometric specimen stage. Received Apr. 5, ”79.Accepted

June 13, ’79.

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M. S. FORBES AND N. SPERELAKIS RESULTS

In the tissue space of mouse heart, fibroblasts can be readily distinguished from neighboring cell types by their lack of a covering of basal lamina (figs. 1-3,8,9).Myocardial fibroblasts are characterized by their extensive thin cytoplasmic processes, which follow a meandering course through the tissue space (fig. 1).The majority of fibroblast processes within mouse heart are unremarkable in terms of their ultrastructure; however, highly-structured fibroblast processes were found in the ventricular myocardium of the six mice used in this study (figs. 1-10).In each of the more than 30 examples examined, a portion of each process was particularly a t t e n u a t e , averaging approximately 40 nm in thickness and ranging from 164-918 nm in length. Inspection of such segments usually revealed the presence of a distinct intracellular electron-opaque line (“central lamina”), approximately 12.5 nm in width, positioned midway between the profiles of the limiting plasma membranes (figs. 1-10]. In no instance did the intracellular laminae of myocardial fibroblasts extend beyond a thickness of more than three consecutive thin sections. We have in fact found series of thin sections in which laminae appeared and then disappeared, either leaving fibroblast profiles which showed cytoplasmic continuity throughout their lengths or revealing profiles in which laminate areas appeared a t a deeper level of the process. We conclude therefrom that such structures are ribbon-like bodies, probably no more than 200 nm in width (fig. 3). Under close scrutiny, bridging structures were sometimes found to be present between the central laminae and the inner surfaces of the limiting cell membranes of some fibroblast processes (figs. 3-7, 8-10). The laminae themselves were seen to be composed either of material arranged into a confluent membrane-like array (figs. 1-4, 6, 8-10) or of single rows of spheroidal or ellipsoidal subunits (figs. 5 , 7, 8 ) . These two configurations could be present in alternate sections of the same lamina (figs. 4,5) or might appear in the same section a t different points along the length of the lamina (fig. 8 ) . Tilting of fibroblast segments did not result in the resolution of separate subunits within examples of the confluent form of lamina. A rather close resemblance was seen be-

tween focal laminate processes of fibroblasts and saccules of myocardial junctional sarcoplasmic reticulum (J-SR) (fig. 91, in that both of these structures contain ordered internal material: the fibroblast process possesses a central lamina (fig. 101, the J-SR an intrasaccular collection of opaque “junctional granules” that often fall into a linear arrangement (fig. 11). DISCUSSION

Focal laminate segments of fibroblast processes in mouse heart closely resemble the thin profiles found in cultured acoustic Schwannoma cells (Conley and Herman, ’73) and in the P t K z rat kangaroo kidney cell line (Franke et al., ’78). The presumed adhesive structures described within all these cells are totally intracellular and therefore are associated with the inner faces of the plasma membranes. They therefore may be distinguished from the “intracytoplasmic” junctions described in heart by Buja et al. (’741, since those structures are produced by the folding over of cells on themselves, which brings the extracellular faces of specialized membranes, such as those of the intercalated discs, to abut one another. The attenuated processes in Schwannoma cells (Conley and Herman, ’731 bear somewhat greater similarity to focal laminate segments of fibroblasts in terms of their dimensions (lengths of 400-600 nm and total estimated thickness of 37 nm) than do the PtK2 segments, which may reach 2 p m in length (Franke et al., ’781, and whose thickness appears to be less than 30 nm. The pronounced central lamina of the fibroblast processes, found in the mouse heart in vivo, is absent from the flattened cell segments of cultured cells, which instead exhibit a distinct septa1 appearance. This appearance is derived from the presence of “thin linear ladder-like formations” (Conley and Herman, ’73) that span the entire intracellular gap, thereby creating structures which resemble the “septate desmosomes” formed intercellularly in a variety of invertebrate and vertebrate tissues (e.g., Rose, ’71; Friend and Gilula, ’72; Sotelo and Llinas, ’72; Noirot-Timothee and Noirot, ‘73; Schwartz, ’73). Although a septate configuration usually is not obvious in focal laminate segments of fibroblasts, the subunits appearing in some laminae (figs. 5,7) resemble those within the intercellular spaces of rat adrenal cell-to-cell contacts (Friend and Gilula, ’72).

INTRACELLULAR ADHESIONS IN FIBROBLASTS

Other intracytoplasmic complexes that have been described include those formed by interaction of an intracellular organelle with the inner side of the plasma membrane or its derivatives. Such complexes include: (a) the subsarcolemmal saccules of sarcoplasmic reticulum (“peripheral” junctional SR) of cardiac muscle (Forbes and Sperelakis, ’77) and skeletal muscle (Spray et al., ’74); (b) interior couplings between junctional SR and transverse tubules in cardiac (Forbes and Sperelakis, ’77) and skeletal muscle cells (Kelly, ’69; Franzini-Armstrong, ’70); (c) the subsurface cisterns of endoplasmic reticulum in neurons (Rosenbluth, ’62; Le Beux, ’72); and (d) the “imaged-desmosomes” of fetal guinea pig myocardial cells, which appear to be composites of desmosomes and SR (Forbes and Sperelakis, ’75). Intraorganellar junctions are found as well: under a variety of conditions, the luminal surfaces of certain portions of skeletal muscle SR collapse upon themselves to form pentalaminar membrane appositions (“zippers”: Wallace and Sommer, ’75; Forbes and Sperelakis, ’79). In mitochondria, linear and septate structures have been discovered in the intermembrane spaces (Saito e t al., ’74; Smith and Klima, ’76). The focal laminate segments of fibroblasts superficially resemble extensive tight junctions (zonulae occludentes). However, close inspection of the segments reveals no fusion of the inner lamellae of the limiting unit membranes. Rather, the central linear opacity which characterizes focal laminate segments is not related to membrane structure, but instead resembles the “central extracellular lamina” found in the intercellular space between the plaques of desmosomes (Rayns et al., ’69). The function of the fibroblast lamina may be similar to that of the external leaflet of the desmosome. That is, the “intracellular lamina” may exert an adhesive action from within the cell, thereby causing the characteristic close apposition of the membranes of these segments. The irregularly-disposed “bridges” (figs. 4, 6, 8) may be the means whereby the narrowing effect is achieved; a similar role is attributed t o the “side arms” associated with the external lamina of desmosomes (Rayns et al., ’69). In focal laminate segments, the bridges appear to emanate from the central lamina, and usually are not oriented in register; hence, they do not extend completely across the intracellular gap as do

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the “rungs” within intracellular septate desmosomes (Conley and Herman, ’73). A “central dense layer” within cardiac junctional SR (J-SR) has been described by Walker e t al. (’70); the opaque structural material there has also been assigned the term “junctional granules” by Sommer and Johnson (‘68). It has been speculated that this intrasaccular material is the framework with which Ca ++,Mg”-dependent ATPase molecules are associated (Forbes and Sperelakis, ’74). In addition, junctional granules may perform a mechanical function. A t the point of transition between the tubules of “free” or “network” SR and the saccules of J-SR there is usually found a pronounced flattening of the SR profile (Forbes and Sperelakis, ’77); a similar change of configuration is found a t the junction between non-laminate and laminate segments of myocardial fibroblast processes (fig. 9). Walker et al. (’70) detected projections from the SR central dense layer that are similar to the bridges between the central lamina and the innermost lamellae of the cell membrane of the myocardial fibroblast (fig. 11). They argue that such connections “exert a holding force,” acting thereby to narrow the J-SR lumen. The degree of parallelism achieved by J-SR limiting membranes is in most instances surpassed by that of the focal laminate segments, probably because of the far more elaborate intraluminal architecture of the latter structures. The focal laminate fibroblast processes present in vivo in mouse heart, together with the intracellular septate desmosomes found in cultured cells (Conley and Herman, ‘73; Franke et al., ’78), form a category of structures that are unique examples of seeming adhesions between the inner plasmalemmal faces of the same cell. It appears that the focal laminate segment is itself unique within this category in two respects. First, it is present within the intact organism; second, it contains an array of subunits distinctly aligned parallel to the planar axis of the cell process, rather than exhibiting a collection of vertically-oriented bodies. It should be noted that focal laminate segments are present (as are the structural variations of their central laminae) after treatment of mouse heart with a variety of aldehyde-based fixatives (MATERIALS AND METHODS). Such modified fibroblasts, furthermore, are not limited in occurrence to mouse heart; we have recently discovered similar

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focal laminate segments in fibroblasts of c a t atrial myocardium (Forbes, unpublished observations). ACKNOWLEDGMENTS

This research was supported in part by American Heart Grant-in-Aid 78-753. We are grateful to Dr. John Rash of t h e Department of Pharmacology and Experimental Therapeutics of t h e University of Maryland School of Medicine for the use of t h e Siemens electron microscope. LITERATURE CITED Bois, R. M. 1973 The organization of the contractile apparatus of vertebrate smooth muscle. Anat. Rec., 177: 61-78. Buja, L. M., V. J. Ferrans and B. J. Maron 1974 Intracytoplasmic junctions in cardiac muscle cells. Am. J. Pathol., 74: 613-648. Conley, F. K., and M. M. Herman 1973 Intracellular sept a t e desmosome-like structures in a human acoustic Schwannoma in uitro. J. Neurocytology, 2: 457-464. Forbes, M. S., and N. Sperelakis 1974 Spheroidal bodies in the junctional sarcoplasmic reticulum of lizard myocardial cells. J. Cell Biol., 60: 602-615. 1975 The “imaged-desmosome”: a component of intercalated discs in embryonic guinea pig myocardium. Anat. Rec., 183: 243-258. 1977 Myocardial couplings: their structural variations in the mouse. J. Ultrastruct. Res., 58: 50-65. 1979 Ruthenium red staining of skeletal and cardiac muscles. Cell and Tissue Research, 200: 367-382. Franke, W. W., C. Grund, E. Schmid and E. Mandelkow 1978 Paracrystalline arrays of membrane-to-membrane cross bridges associated with the inner surface of plasma membrane. J. Cell Biol., 77: 323-328. Franzini-Armstrong, C. 1970 Studies of the triad. I. Structure of the junction in frog twitch fibers. J. Cell Biol., 47: 488-499. Friend, D. S., and N. B. Gilula 1972 A distinctive cell contact in the rat adrenal cortex. J. Cell Biol., 53: 148-163. Kelly, D. E. 1969 The fine structure of skeletal muscle triad junctions. J. Ultrastruct. Res., 29: 37-49. Le Beux, Y. J. 1972 Subsurface cisterns and lamellar bodies: particular forms of the endoplasmic reticulum in the neurons. Z. Zellforsch. mikrosk. Anat., 133: 327-352. McNutt, N. S., and R. S. Weinstein 1970 The ultrastructure of the nexus. A correlated thin-section and freezecleave study. J. Cell Biol., 47: 666-688. Melax, H., and T. S. Leeson 1972 Electron microscope study

of myocardial tissue space contents in r a t heart. Cardiovasc. Res., 6: 89-94. Millonig, G. 1962 Further observations on a phosphate buffer for osmium solutions in fixation. Proc. Congr. Electron Microsc. 5th, 1962, Vol. 2: P-8. Noirot-Timothee, C., and C. Noirot 1973 Jonctions et contacts intercellulaires chez les insectes. I. Les jonctions septkes. J. de Microscopie, 17: 169-184. Rayns, D. G., F. 0. Simpson and J. M. Ledingham 1969 U1trastructure of desmosomes in mammalian intercalated disc; appearance after lanthanum treatment. J. Cell Biol., 42: 322-326. Rose, B. 1971 Intercellular communication and some structural aspects of membrane junctions in a simple cell system. J. Membrane Biol., 5: 1-19. Rosenbluth, J. 1962 Subsurface cisterns and their relationship to the neuronal plasma membrane. J. Cell Biol., 13: 405-421. Rostgaard, J., and 0.Behnke 1965 Fine structural localization of adenine nucleoside phosphatase activity in the sarcoplasmic reticulum and the T system of rat myocardium. J. Ultrastruct. Res., 12: 579-591. Saito, A,, M. Smigel and S. Fleischer 1974 Membrane junctions in the intermembrane space of mitochondria from mammalian tissues. J. Cell Biol., 60: 653-663. Schwartz, W. J. 1973 A septate-like contact in the rat retina. J. Neurocytology, 2: 85-89. Smith, M. N., and M. Klima 1976 Incidence of intermembrane alterations in human heart mitochondria: a preliminary ultrastructural study. Am. Heart J., 91: 563-570. Sommer, J. R., and E. A. Johnson 1968 Cardiac muscle. A comparative study of Purkinje fibers and ventricular fibers. J. Cell Biol., 36: 497-526. Sommer, J. R., N. R. Wallace and W. Hasselbach 1978 The collapse of the sarcoplasmic reticulum in skeletal muscle. 2.Naturforsch., 33: 561-573. Sotelo, C., and R. L l i n L 1972 Specialized membrane junctions between neurons in the vertebrate cerebellar cortex. J. Cell Biol., 53: 271-289. Spray, T. L., R. A. Waugh and J. R. Sommer 1974 Peripheral couplings in adult vertebrate skeletal muscle. Anatomical observations and functional implications. J. Cell Biol., 62: 223-227. Venable, J. H., and R. Coggeshall 1965 A simplified lead citrate stain for use in electron microscopy. J. Cell Biol., 25: 407-408. Walker, S. M., G. R. Schrodt and M. B. Edge 1970 Electrondense material within sarcoplasmic reticulum apposed to transverse tubules and to the sarcolemma in dog papillary muscle fibers. Am. J. Anat., 128: 33-44. Wallace, N., and J. R. Sommer 1975 Fusion of sarcoplasmic reticulum with ruthenium red. Proc. 33rd Annu. Meeting EMSA, pp. 500-501.

PLATES

PLATE 1 EXPLANATION OF FIGURES

1 Left ventricular wall of mouse myocardium. A slender process of a fibroblast (FP)is inserted in the interstice (“tissue space”) between two transversely-sectioned cardiac muscle cells (MC). A prominent feature of the fibroblast process is the presence of a particularly narrow segment along part of its profile (between arrows). This attenuate segment, approximately 0.92 p m in length, is characterized by a n opaque intracytoplasmic line (fig. 2), which gives the fibroblast process a t this level a multilayered or laminated appearance. X 34,500. 2 Detail of t h e laminate fibroblast segment shown in figure 1. At all points along t h e

segment, the limiting membranes on either side of t h e centrally-located lamina exhibit a close, parallel relationship to one another as well as to t h e lamina. Note that basal laminar material is not present a t the outer surface of the fibroblast, although a distinct cell coat is associated with the adjacent muscle cells. x 94,000.

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INTRACELLULAR ADHESIONS IN FIBROBLASTS M. S. Forbes and N. Sperelakis

PLATE 1

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PLATE 2 EXPLANATION OF FIGURES

3

Left ventricular wall. A laminate segment of a fibroblast process is sandwiched between a cardiac muscle cell (MC) and another fibroblast (F). This laminar profile is particularly short (0.185 Fm), and may be an end-on view of a lamina whose length is far greater than its width. x 68,000.

4

Detail of the laminate segment shown in figure 3. The central lamina cannot be resolved a t any point into a unit membrane, although the trilaminar structure of the cell membrane profiles (CM) is obvious in this section. At several points, projections (arrows) extend between the lamina and the inner surface of the adjacent cell membrane. x 220,000.

5 Another section of the laminate segment shown in figures 3 and 4. At this level, the lamina seems to be composed of a series of ellipsoidal bodies (asterisks), which gives it a scalloped appearance. x 220,000. 6

Laminate fibroblast process found in 3 periarterial space in right ventricle. Numerous perpendicularly-oriented “bridges” are present (arrows) between the lamina and the cell membranes. x 220.000.

7 Laminate fibroblast process in which the lamina does not appear a s a single continuous structure, but instead is composed of discrete bodies that in profile are ellipsoidal or circular. At several points (arrows), these subunits of the lamina appear to be connected to t h e inner cell membrane surfaces. x 220,000, 8 The left-hand portion of this laminate segment of a fibroblast contains a distinct, confluent lamina with cross-bridges (arrows); in contrast, the rightmost laminar region is made up of discrete, linearly-arranged particles (asterisks). x 153,500.

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INTRACELLULAR ADHESIONS IN FIBROBLASTS M. S. Forbes and N. Sperelakis

PLATE 2

PLATE 3 EXPLANATION OF FIGURES

9 A laminate fibroblast segment, shown for the comparison of its morphology with t h a t of a saccule of junctional sarcoplasmic reticulum (J-SR) located a t the periphery of t h e adjacent myocardial cell. Note the abrupt narrowing of the profile of t h e fibroblast a t the junction between its nonlaminate and laminate portions. x 109,000.

10 Detail of the fibroblast in figure 11. I t s central lamina averages 11.4 nm in width, and the total thickness of the process is ca. 45 nm. X 272,000. 11 A portion of the J-SR saccule shown in figure 11. The overall thickness of the saccule is somewhat less (ca. 29-37 nm) than t h a t of the laminate fibroblast segment (cf. fig. lo), and its internal opaque contents (“junctional granules”) are less highly-structured, although in some regions they can be resolved into linear structures (arrows) having a width of 7-8 nm. Some of these structures appear to contact the inner surface of the limiting membrane of the saccule ( p i n t s of contact marked by the asterisk). The bodies (‘‘junctional processes”; arrowheads) t h a t lie in t h e space between t h e J-SR and the inner face of t h e sarcolemma (SL) may be evaginations of the J-SR membrane. X 272,000.

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PLATE 3

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Focal laminate segments in cytoplasmic processes of mouse myocardial fibroblasts.

Focal Laminate Segments in Cytoplasmic Processes of Mouse Myocardial Fibroblasts M. S. F O R B E S AND N. S P E R E L A K I S Department of Physiology...
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