High-resolution scanning electron microscopic studies on the three-dimensional structure of the transverse-axial tubular system sarcoplasmic reticulum and intercalated disc of the rat myocardium.код для вставкиСкачать
THE ANATOMICAL RECORD 228:277-287 (1990) High-Resolution Scanning Electron Microscopic Studies on the Three-Dimensional Structure of the Transverse-Axial Tubular System, Sarcoplasmic Reticulum and Intercalated Disc of the Rat Myocardium TAKURO OGATA AND YUICHI YAMASAKI Department of Surgery, Kochi Medical School, Kochi, Japan ABSTRACT The three-dimensional structure of the transverse-axial tubular system, sarcoplasmic reticulum (SR), and intercalated disc of the rat left ventricle was examined by high-resolution scanning electron microscopy after removal of the cytoplasmic matrices by the osmium-DMSO-osmium procedure. In the intermyofibrillar space, the transverse tubules (T-tubules) are accompanied by longitudinally oriented axial tubules and together form a transverse-axial system. The junctional SR is usually small but occasionally medium or large in size and couples with the T- or with the axial tubules. On the surface of the junctional SR facing the T- or the axial tubule, tiny junctional processes are seen. One or two sarcotubules, the so-called Z-tubules, frequently run parallel to the T-tubule. The sarcotubules derived from the junctional SR or from the Z-tubule run longitudinally or obliquely and form polygonal meshes around the myofibrils. On the surface of the SR a t the H-band level, small fenestrations of 12-40 nm in diameter, and tiny hollows 8-20 nm in diameter are seen. Bulbous swellings of the SR, the corbular SR, are preferentially seen near the Z-band. The large and flat SR, known as the cisternal SR, intercalates among the SR meshes. In the subsarcolemmal space, the sarcotubules form a multilayered network (peripheral SR). The cisternal SR is frequently intercalated in these meshes and closely associated with the inner surface of the sarcolemma. The intercalated disc appears a s a prominently undulated membrane demarcating the border between two adjacent heart muscle cells, and occasionally small projections 60-90 nm in diameter and 200-600 nm in length display on its surface. Three-dimensional models of the mammalian cardiac muscle fiber based on transmission electron microscope (TEM) observations of thin, sometimes serial, sections have been proposed by some investigators (Porter and Palade, 1957; Fawcett and McNutt, 1969; Bossen et al., 1978). The three-dimensional organization of the transverse-axial tubular system (TATS) and the sarcoplasmic reticulum (SR) has been examined in freeze-fracture replicas (Scales, 1981; Forbes e t al., 1984; Forbes and Van Niel, 1988), and by high-voltage electron microscopy after labeling with horseradish peroxidase and lanthanum (Sommer and Waugh, 1976), by osmium-ferrocyanide postfixation method (Segretain e t al., 1981; Forbes and Van Niel, 1988) and by the Golgi black reaction method (Scales, 1983). Using the osmium-DMSO-osmium procedure (Tanaka and Naguro, 1981),the three-dimensional structure of the membrane system of the myocardium of the rat (Ohmori, 1984) and of the dog (Yoshikane et al., 1986) were directly observed by scanning electron microscopy (SEM). However, in these studies, the specimens were coated with metal, and consequently the details of the surface structure of the membrane were concealed un0 1990 WILEY-LISS, INC. der the metal coating. In the present study, the membrane system of the rat ventricle was exposed by the osmium-DMSO-osmium procedure and impregnated with osmium by the tannic acid-osmium staining (Murakami, 1973), followed by osmium-hydrazine (Kubotsu and Ueda, 1980). The specimens were examined by high-resolution SEM on a Hitachi S-900 with a resolving power of 7 A without metal coating, and the detailed three-dimensional structure of the TATS, SR, and intercalated discs was elucidated. MATERIALS AND METHODS Male Wistar rats weighting 250-300 g were used. The methods employed in the present study are the same as described in the previous paper (Ogata and Yamasaki, 1987). In short, the left ventricle was fixed with 0.5% paraformaldehyde-1 .O% glutaraldehyde so- Received October 23, 1989; accepted April 23, 1990. Address reprint requests to Prof. Takuro Ogata, Department of Surgery, Kochi Medical School, Nankoku, Kochi, 783, Japan. 278 T. OGATA AND Y. YAMASAKI lution in 0.067 M cacodylate buffer (pH 7.4) for 15 minutes by retrograde perfusion through the thoracic aorta. The muscle was cut into small pieces and further fixed for 15 minutes in the same fixative. The tissue blocks were immersed in dimethyl sulfoxide (DMSO) and frozen in liquid propane cooled with liquid nitrogen. The specimens were cracked with a single-edged razor blade by striking it with a hammer in a freezecracking apparatus Eiko TF-1. Then they were postfixed with 1%OsO, in 0.067 M cacodylate buffer (pH 7.4) for 1 hour and left standing in 0.1% Os04 in the same buffer at 20°C for 72-96 hours to remove the cytoplasmic matrix (Tanaka and Naguro, 1981).Tissue specimens prepared in this way were impregnated with osmium by the conductive staining method (Murakami, 1973). Afterwards they were dehydrated in ethanol and dried in a Hitachi HCP-1 critical-point dryer. The dried specimens were mounted on a specimen stub and impregnated again with osmium by the osmium-hydrazine method (Kubotsu and Ueda, 1980). The observation was done under a high-resolution, field-emission type SEM, the Hitachi S-900. RESULTS The osmium-DMSO-osmium method (Tanaka and Naguro, 1981) used in the present study proved to be very useful in disclosing the architecture of the intercellular membranous structures. Myofilaments and cytoplasmic matrices were removed by maceration in the Os04 solution, and the membranous structures could be disclosed without introducing any significant artifact. The sarcolemma appeared as a fairly smooth membrane surrounding the muscle fiber, with numerous caveolae 70-110 nm in diameter attached to its external and internal surfaces (Figs. 1, 16a). Large-diameter invaginations from the sarcolemma extended deeply into the interior of the muscle fiber and transformed into the transverse tubules (T-tubules) (Fig. 1). The T-tubules ran transversely to the long axis of the muscle fiber a t each Z-band level and made relatively frequent interconnections with the axial tubules, which usually ran parallel to the long axis of fiber (Figs. 2c, 6), although some were obliquely oriented (Fig. 2a). The interconnecting point of the T- and axial tubules was distended (Fig. 7). The diameter of the T-tubules ranged between 70 and 100 nm. Some of them were therefore thicker than those of the sarcotubules (Fig. 6), which ranged between 50 and 80 nm, but others (Figs. 3,4) were of almost the same diameter as A bbreuiations A C D H I J M S T Z ZT * axial tubule corbular SR desmosome H-band level intercalated disc junctional SR mitochondria sarcolemma T-tubule Z-band level Z-tubule cisternal SR the sarcotubules. The diameter of the axial tubules ranged between 90 and 120 nm (Figs. 2c, 7); their average diameter was therefore slightly thicker than that of the T-tubules. The T- and axial tubules usually appeared as slender tubules with almost the same diameter along their length (Fig. 2a, c), but with occasional swellings (Figs. 7, 9). In addition, bell-shaped or wartlike swellings were occasionally seen, and some coupled with the junctional SR (Fig. 4). The SR formed lace-like networks inside the fiber (Fig. 6). At the Z-band level, the myofibrils were encircled by sarcotubules. These sarcotubules were first observed by TEM and have been called the Z-tubules (Simpson and Rayns, 1968; Forbes and Sperelakis, 1980). Most often, the Z-tubules lay closely adjacent to the T-tubule (Figs. 3, 6, 8). In SEM images, the Z- and T-tubule were arranged parallel to each other, but usually in slightly different planes (Figs. 6, 8). The T-tubule was either sandwiched between two Z-tubules (Fig. 8) or accompanied by one Z-tubule only (Figs. 3, 6). Numerous tiny projections 10-16 nm in diameter and 6-12 nm in height were arranged 25 nm apart from each other on the surface of the Z-tubule facing the T-tubule. These projections seem to correspond to the feet (Franzini-Armstrong, 1970) or junctional processes (Sommer and Johnson, 1979) already observed by TEM. Such arrangements were seen a t irregular intervals and did not occur throughout the entire length of the Z-tubule (Fig. 8). Similar tiny projections were occasionally observed on the surface of the T-tubule (Figs. 8, 12a),but they were far fewer and lacked regularity of arrangement. The junctional SR that coupled with the T-tubule was usually small and flat (Figs. 3, 9), although medium-sized (Fig. 10) or large-sized junctional SR (Fig. 11)was also seen. The coupling of the SR to the axial tubules was also demonstrated in SEM (Fig. 2) and Fig. 1. SEM image of the transformation of the sarcolemma into the T-tubule. a: Large-diameter invaginations of the sarcolemma extend deeply into the fiber at the Z-band level and transform into the Ttubules. Numerous caveolae (arrows) are attached to the external and internal surfaces of the sarcolemma. x 21,000. b Higher magnification of part of a from a slightly different angle. The initial portion of the T-tubule is fractured, and its inner structure is exposed (black star). Arrows show caveolae. x 69,000. Fig. 2. SEM images of the axial tubules. a: The axial tubules run obliquely and connect with the T-tubules. Arrows show the coupling of the junctional SR to the axial tubules. x 45,000. b A section of a. Junctional SR couples with the interconnecting part of the axial tubule and the T-tubule. Arrow shows polyribosomes. x 109,000, c: Longitudinally running axial tubule. Note that the diameter is fairly uniform throughout its course. x 34,000. Fig. 3. SEM image of the Z-band region. The Z-tubule runs parallel to the T-tubule. Small junctional SR couples with the T-tubule. Note the small size of the SR mesh a t the Z-band level. x 78,000. Fig. 4. A bell-shaped swelling of the T-tubule is coupled by junctional SR. The ovoid corbular SR attaches to the sarcotubule. x 91,000. Fig. 5. TEM micrograph showing the coupling of the junctional SR to the axial tubule (arrows). x 41.000. MEMBRANE SYSTEM OF THE HEART Figs. 1-5. 279 280 T. OGATA AND Y. YAMASAKI Figs. 6-10. 281 MEMBRANE SYSTEM OF THE HEART TEM pictures (Fig. 5). SEM images of the transversely fractured T-tubule and junctional SR complex showed that, usually, the T-tubule was encircled by the junctional SR only partially (Fig. 12a), although occasionally completely encircled structures were also seen (Fig. 12b).Tiny feet were also seen on the surface of the junctional SR facing the T-tubule (Fig. 12a). Slender sarcotubules arising from the junctional SR or from the Z-tubule ran longitudinally or obliquely toward adjacent sarcomeres at both sides and formed polygonal meshes of various sizes. These meshes were small, i.e., 0.1-0.3 pm in diameter, and oval at the I-band (Figs. 3, 6), whereas a t the A-band they were larger, i.e., 0.3-0.5 Fm maximal length, and long-ellipsoid shaped (Figs. 6, 15). The sarcotubules were fairly uniform in diameter throughout their course, except for occasional distentions (Fig. 151, and presented a relatively smooth surface. The plate-like SR, i.e., the cisternal SR, was relatively frequently intercalated among the SR meshes (Figs. 6,13a). This structure was preferentially located at the I-band level, but occasionally it was also seen at the A- and the H-band levels (Fig. 13b). Polyribosomes were frequently attached on its surface (Fig. 13). Polyribosomes were also sporadically seen on the surface of the junctional SR (Fig. 2b) and sarcotubules (Fig. 13b). At the H-band level, the sarcotubules rather often formed small meshes or fenestrations (12-40 nm in diameter), which passed through the SR (Fig. 151, and their surface exhibited a few tiny hollows (8-20 nm in diameter), which did not seem to pass through the SR (Fig. 15). These hollows were also observed on the surface of the cisternal SR, which was occasionally present at the H-band level (Fig. 13b). Bulbous terminal swellings of the SR 80-170 nm in diameter, i.e., the corbular SR, were preferentially seen near the Z-band (Figs. 4,6,14). Some appeared as hemispheric swellings on the surface of the sarcotubules (Fig. 14a), but most were spherical or ovoid and connected to the sarcotubules (Figs. 4,14b, c). The surface of the corbular SR was either smooth (Figs. 4,14b) or provided with tiny granular projections about 8 nm in diameter (Fig. 14c), which resembled the feet of the junctional SR. In the intermyofibrillar space, numerous spherical or ovoid mitochondria were accumulated (Fig. 6). Around the mitochondria, the SR was less well developed than around the myofibrils. Simple SR networks transversely crossed the mitochondria at the Z- and H-band levels, and a few sarcotubules originating from them ran longitudinally at the I- and the A-band and connected the SR network of the Z-band with that of the H-band (Fig. 6). The portion of the mitochondria underlying the SR was frequently constricted (Fig. 12a). In the subsarcolemmal space, the peripheral (subsarcolemmal) SR formed polygonal meshes 80-200 nm in diameter (Fig. 16). These meshes were not arranged in a single plane, but rather formed multilayered networks (Fig. 16). Plate-like cisternal SR, polygonal in shape and 130-400 nm in diameter, was frequently intercalated among these meshes (Fig. 16). Most of these subsarcolemmal cisternal SR were closely associated with the inner surface of the sarcolemma (Fig. 16).Polyribosomes were frequently attached to the surface of the subsarcolemmal junctional SR (Fig. 16b). Numerous spherical caveolae were also seen on the surface of the sarcolemma and frequently interspersed in the SR meshes (Fig. 16). Very thin tubules with beaded structures (about 20 nm in diameter) were occasionally seen among the peripheral SR network (Fig. 16b). The sarcolemma was deeply invaginated between two adjacent cardiac cells and transformed into the intercalated disc (Fig. 17).The intercalated disc appeared as a markedly undulated membrane bordering two cardiac cells (Figs. 17, 18). At higher magnification, its surface was fairly smooth (Fig. 18) with only a few scattered caveolae (Fig. 19a, c). These caveolae were also seen on the surface of the intercalated disc by TEM (Fig. 20a). Slender projections about 60-90 nm in diameter and 200-600 nm in length occasionally protruded from the intercalated disc (Fig. 19b). Some were twisted (Fig. 19b).In TEM micrographs, they were surrounded by membrane, and their inside was filled with amorphous substance (Fig. 20b). Frequently, the SR was closely associated with the surface of the intercalated disc (Fig. 21). Some of the SR had a cisternal-like appearance (Fig. 21). Occasionally the summits of the intercalated disc were surrounded by the SR (Fig. 22). DISCUSSION Fig. 6. SEM image of the SR network. T-tubules run transversely at the Z-band level. The axial tubule connects with a T-tubule a t both ends. Note that the diameter of these tubules is larger than that of the sarcotubules. Ovoid mitochondria arrange themselves in the intermyofibrillar spaces. Around the mitochondria, the SR forms simple meshes a t the H- and Z-band levels. At the level of the A-band, a few longitudinally arranged sarcotubules are seen. x 30,000. Fig. 7. Higher magnification of a part of Figure 6. Note small (small arrow) and large (large arrow) wart-like swellings of the axial tubule. x 106,000. Fig. 8. The T-tubule is sandwiched between two Z-tubules. Periodically arranged tiny feet (arrowheads) are seen on the surface of the 2-tubule facing the T-tubule. Occasionally, a small projection of approximately the same size (arrow) is seen on the surface of the Ttubule. The inside of the 2-tubule is partly exposed. x 113,000. Fig. 9. Swellings of the T-tubule (arrows). x 86,000. Fig. 10. The medium-sized junctional SR couples with the T-tubule. x 91,000. In the mammalian heart, the T-tubules are often accompanied by longitudinally oriented axial tubules and together form the transverse-axial tubular system usually abbreviated as TATS (Sperelakis and Rubio, 1971; Forbes et al., 1984). In the present study, the TATS was clearly demonstrated by SEM. In the mouse ventricle, the axial tubules have been reported frequently to run obliquely (Forbes et al., 1984), and in the present investigation a similar orientation was observed in the rat. Occasional swellings of the T-tubule were observed, and some were coupled with SR. Similar swellings had been observed in the rat ventricle using the ferrocyanide-osmium method (Tomita and Ferrans, 1987). In the dog ventricle, the SR appears as a tight network of tubules in the region of the H- and Z-bands and forms larger meshes at the A- and I-band levels (Yoshikane et al., 1986). The present results showed that the 282 T. OGATA AND Y. YAMASAKI Figs. 11-1 5. MEMBRANE SYSTEM OF THE HEART SR of the rat ventricle displays a similar distribution. Under SEM, most of the Z-tubules were demonstrated to run also in parallel to the T-tubule. Until now, the relationship between the Z- and the T-tubule has not been clearly described in the literature, probably because of the difficulty in obtaining sufficiently long profiles of both tubules in TEM sections. In the present SEM study, numerous tiny projections closely resembling in location, size, and periodicity the tiny feet (Ferguson et al., 1984) or junctional processes, which are considered to be the apparatus for coupling of the SR to the T-tubule (Rayns et al., 1975; Forbes and Sperelakis, 1977; Sommer and Johnson, 1979), were seen on the surface of the Z-tubule facing and T-tubule and on the surface of the junctional SR exposed to the TATS. These findings suggest that the excitation-contraction coupling occurs not only between the junctional SR and the TATS (Sommer and Johnson, 19791, but also between the Z- and T-tubule. The coupling of the Z-tubule to the T-tubule has not been reported yet, probably because i t is difficult to discriminate the junctional SR from the Z-tubule by TEM. Similar tiny projections were occasionally seen on the surface of the T-tubule, but they are far fewer and lack regularity of their arrangement. The nature of these projections is, however, unknown. In the SEM images of the transversely fractured Ttubule and junctional SR complex, the junctional SR, most frequently, encircled the T-tubule only partly, but complete encircling of the T-tubule by the junctional SR was occasionally seen, as already reported by TEM (Sommer and Johnson, 1969; Forbes and Sperelakis, 1977). The coupling of the junctional SR to the axial tubule already reported in the dog myocardium (Forbes and Van Niel, 1988) was also observed by SEM and TEM in the present study. Numerous tiny fenestrations in the H-band collar were demonstrated in the rabbit myocardium by the Golgi black reaction method (Scales, 1983) and in the Fig. 11. Two large junctional SR with cisterns-like appearance coupling with T-tubules. x 44,000. Fig. 12. Transversely fractured T-tubule and junctional SR. a: The junctional SR partly encircles the T-tubule. On the surface of the SR facing the T-tubule, tiny feet (arrowheads) are arranged a t 25 nm intervals. Occasionally, a solitary tiny projection (arrow) is seen on the surface of the T-tubule. Note the constriction of the mitochondrion beneath the sarcotubules. x 113,000. b Junctional SR completely encircling the T-tubule. x 113,000. Fig. 13. SEM images of the cisternal SR. a: Cisternal SR at the I-band level. Arrows show polyribosomes. X 113,000. b Cisternal SR at the H-band level. Note tiny hollows (arrowheads) on its surface. Arrow shows polyribosomes attached on the surface of the sarcotubules. x 117,000. Fig. 14. SEM images of corbular SR. a: The corbular SR appears as a hemispherical swelling. x 113,000. b: Ovoid-shaped corbular SR with smooth surface. ~ 5 7 , 0 0 0 .c: Spherical corbular SR with tiny projections (arrowheads) on its surface. Similar projections (arrows) are seen on the surface of a low, round elevation of the sarcotubule. x 113,000. Fig. 15. Higher magnification of the SR network. Sarcotubules form large size meshes at the level of the A-band (Ab).At the H-band level, a small mesh (small arrow) and tiny hollows (arrow heads). are seen. Large arrow shows distention of the sarcotubule. x 113,000. 283 guinea pig myocardium by the osmium-ferrocyanide postfixation method (Forbes and Van Niel, 1988). An early TEM study on Ambystoma muscle described smaller circular patches (20 nm) in the H-band collar, which were referred to as pores or thin places in the membrane (Porter and Palade, 1957) and later were thought to represent perforations in one of the two membranes of this collar (Franzini-Armstrong, 1963). Further TEM studies on the SR of the frog skeletal muscle demonstrated that they were fenestrations right through the H-band collar (Peachey, 1965). However, SEM observations on the H-band collar of the frog muscle revealed that there are fenestrations of 20-50 nm diameter and tiny membrane depressions (15-20 nm in diameter) that seem not to pass completely through the H-band collar and were tentatively named H-band hollows (Ogata and Yamasaki, 1987). In the present observations, similar fenestrations and hollows were observed in the H-band collar of the cardiac muscle. The nature of these hollows, however, is still unknown. Corbular SR is found attached primarily to the perimyofibrillar SR network, but not to the subsarcolemma1 SR arrays (Forbes and Van Niel, 1988), and is preferentially located at the Z-band level. From highvoltage electron microscopic studies, the membranous profiles of the corbular SR are always continuous with the endoplasmic reticulum (Sommer and Waugh, 1976). The continuity of the corbular SR to the sarcotubules was also observed by SEM. The function of the corbular SR is, however, unknown. Immunocytochemically, it was shown that calsequestrin, which has been proposed to store Ca2+ in the lumen of the SR in resting muscle, is confined to the lumen of the junctional SR and of the corbular SR (Jorgensen and McGuffee, 1987). It is interesting to note that tiny feet-like structures, similar to the feet on the surface of the junctional SR facing the T-tubule, are also seen on the surface of some corbular SR. These tiny projections of the corbular SR have also been reported in the rabbit (Dolber and Sommer, 1984) and in the guinea pig myocardium (Forbes and Van Niel, 1988). The plate-like SR, i.e., the cisternal SR, is relatively frequently intercalated among the SR meshes. On the surface of the cisternal SR, polyribosomes are frequently seen. The presence of ribosomes on the surface of cisternal SR has already been reported (Forbes and Fig. 16 (overleaf).SEM image of the peripheral SR. a: This slightly oblique view shows the sarcotubules forming multilayered polygonal meshes. Large plate-like cisternal SR are intercalated among them. Numerous caveolae (arrows) attach to the surface of the sarcolemma. x 45,000. b Frontal view of the subsarcolemmal SR. The SR forms polygonal meshes. The cisternal SR attaches to the inner surface of the sarcolemma. Arrowhead shows polyribosomes attached on the surface of the cisternal SR. Note that the sarcotubules form multilayered networks (large arrow). Small arrow shows the caveolae interspersed in the SR meshes. Double arrows show a very thin tubule with beaded structure. x 77,000. Fig. 17. Low magnification SEM image of an intercalated disc. The intercalated disc appears as a prominently undulated membrane continuing to the sarcolemma. x 24,000. Fig. 18. At higher magnification, the surface of the intercalated disc is relatively smooth. x 29,000. 284 T. OGATA AND Y. YAMASAKI Figs. 16-18. MEMBRANE SYSTEM OF THE HEART Figs. 19-22. 285 286 T. OGATA AND Y. YAMASAKI Van Niel, 1988). Polyribosomes are also occasionally observed on the surface of the junctional SR and of the sarcotubules. In the rat ventricle, the peripheral (subsarcolemmal) SR is very extensively distributed on the inner surface of the sarcolemma. Similar observations have also been made in the dog ventricle by SEM (Yoshikane et al., 1986). The coupling of the peripheral SR to the sarcolemma has been reported in the rat (Fawcett and McNutt, 1969), in the mouse (Forbes and Sperelakis, 1977), and in the guinea pig (Forbes and Van Niel, 1988). TEM studies showed that the mouse junctional SR saccules (the occasionally expanded regions of the SR containing opaque material within the distended portion) couple with the inner surface of the sarcolemma and interlink with the subsarcolemmal tubular meshwork of the SR network with electron-lucent lumina (Forbes and Sperelakis, 1977). The present SEM observations revealed that the peripheral SR is composed of multilayered polygonal meshes frequently intercalated with polygonal cisternal SR and that most cisternal SR closely attaches to the inner surface of the sarcolemma, whereas the tubular segments are rather apart from it. Judging from these findings, there seems to be little doubt that the cisternal parts of the peripheral SR correspond to the peripheral junctional SR. Fine beaded tubules are occasionally seen among the peripheral SR. The true nature of these fine tubules is, however, unclear. The intercalated disc appears under SEM as a prominently undulated membrane bordering between two cardiac cells. When observed under TEM, it contains numerous intermediate junctions, desmosomes, and gap junctions (Forbes and Sperelakis, 1985). In SEM specimens prepared by the osmium-DMSO-osmium method, the filamentous materials of these junctions are removed by maceration, and therefore the surface of the intercalated disc appears fairly smooth. A few caveloae are seen scatterd on the surface of the intercalated disc, but their number is much smaller than that on the surface of the sarcolemma. The existence of caveolae on the surface of the intercalated disc has not been reported yet. Tiny slender projections are occasionally seen on the surface of the intercalated disc, but their nature awaits further study. Under SEM, the SR was frequently seen in close association with the surface of the intercalated disc, as already reported for TEM studies (Forbes and Sperelakis, 1977). Fig. 19. a: Intercalated disc with a few caveolae (arrowheads) and slender projections (arrows). x 20,000. b: Higher magnification of a part of Fig. 19a. Note that one of the slender projections (arrow) has a club-like appearance and the other two (arrowheads) are twisted. x 51,000. c: Higher magnification of the caveolae (arrowheads) on the surface of the intercalated disc. x 52,000. Fig. 20. TEM micrograph of the intercalated disc. a: Caveolae (arrowheads) on the surface of the intercalated disc. x 46,000. b Clublike projection filled with amorphous material (arrow). x 46,000. Fig. 21. Cisternal SR closely associated with the surface of the intercalated disc. x 65,000. Fig. 22. The SR surrounds the summit of the intercalated disc (arrows). x 76,000. ACKNOWLEDGMENTS The authors are grateful to Dr. K. Nagatani, Hitachi Ltd., for making a n S-900 available; to Messrs. M. Yamada and T. Suzuki, Hitachi Ltd., for taking ultrahigh-resolution SEM pictures; to Ms. K. Ikeda, M. Matsumoto, H. Okazaki, and M. Miyata for their technical assistance throughout this study. LITERATURE CITED Bossen, E.H., J.R. Sommer, and R.A. Waugh 1978 Comparative stereology of the mouse and finch left ventricle. Tissue Cell, 10; 773-784. Dolber, P.C., and J.R. Sommer 1984 Corbular sarcoplasmic reticulum of rabbit cardiac muscle. J . Ultrastruct. Res., 87t190-196. Fawcett, D.W., and N.S. McNutt 1969 The ultrastructure of the cat myocardium, I. Ventricular papillary muscle. J. Cell Biol., 42: 1-45. Ferguson, D.G., H.W. Schwartz, and C. Franzini-Armstrong 1984 Subunit structure ofjunctional feet in triads of skeletal muscle: A freeze-drying, rotary-shadowing study. J. Cell Biol., 99: 1735-1742. Forbes, M.S., and N. Sperelakis 1977 Myocardial couplings: Their structural variations in the mouse. J . Ultrastruct. Res., 58t5065. Forbes, M.S., and N. Sperelakis 1980 Structures located at the levels of the Z bands in mouse ventricular myocardial cells. Tissue Cell, 12t467-489. Forbes, M.S., and N. Sperelakis 1985 Intercalated discs of mammalian heart: A review of structure and function. Tissue Cell, 17: 605-648. Forbes, M.S., and E.E. Van Niel 1988 Membrane systems of guinea pig myocardium: Ultrastructure and morphometric studies. Anat. Rec., 22Zt362-379. Forbes, M.S., L.A. Hawkey, and N. Sperelakis 1984 The transverseaxial tubular system (TATS) of mouse myocardium: Its morphology in the developing and adult animal. Am. J. Anat., 170t143162. Franzini-Armstrong, C. 1963 Pores in the sarcoplasmic reticulum. J. Cell Biol., 19~637-641. Franzini-Armstrong, C. 1970 Studies of the triad. I. Structure of the junction in frog twitch fibers. J . Cell Biol., 47:488-499. Jorgensen, A.O., and L.J. McGuffee 1987 Immunoelectron microscopic localization of sarcoplasmic reticulum proteins in cryofixed, freeze-dried, and low temperature-embedded tissue. J . Histochem. Cytochem., 35t723-732. Kubotsu, A., and M. Ueda 1980 A new conductive treatment of the specimen for scanning electron microscopy. J . Electron Microsc., 29t45-53. Murakami, T. 1973 A metal impregnation method of biological specimens for scanning electron microscopy. Arch. Histol. Jpn., 35: 323-326. Ohmori, T. 1984 Three-dimensional architecture of sarcotubules of rat skeletal and heart muscle cells observed by scanning electron microscopy. (in Japanese) J. Yonago Med. Assoc., 35t241-251. Ogata, T., and Y. Yamasaki 1987 High-resolution scanning electronmicroscopic studies on the three-dimensional structure of mitochondria and sarcoplasmic reticulum in the different twitch muscle fibers of the frog. Cell Tissue Res., 250t489-497. Peachey, L.D. 1965 The sarcoplasmic reticulum and transverse tubules of the frog’s sartorius. J . Cell Biol., 25t209-231. Porter, K.R., and G.E. Palade 1957 Studies on the endoplasmic reticulum. 111. Its form and distribution in striated muscle cells. J. Biophys. Biochem. Cytol., 3t269-300. Rayns, D.G., C.E. Devine, and C.L. Sutherland 1975 Freeze fracture studies of membrane systems in vertebrate muscle. I. Striated muscle. J. Ultrastruct. Res., 50r306-321. Scales, D.J. 1981 Aspects of the mammalian cardiac sarcotubular system revealed by freeze fracture electron microscopy. J. Mol. Cell Cardiol., 13t373-380. Scales, D.J. 1983 111. Three-dimensional electron microscopy of mammalian cardiac sarcoplasmic reticulum at 80 kV. J. Ultrastruct. Res., 83t1-9. Segretain, D., A. Rambourg, and Y. Clermont 1981 Three dimensional arrangement of mitochondria and endoplasmic reticulum in the heart muscle fiber of the rat. Anat. Rec., 200t139-151. Simpson, F.O., and D.G. Rayns 1968 The relationship between the transverse tubular system and other tubules at the 2 disc levels of myocardial cells in the ferret. Am. J. Anat., 122t193-208. MEMBRANE SYSTEM OF THE HEART Sommer, J.R., and E.A. Johnson 1969 Cardiac muscle. A comparative ultrastructural study with special reference to frog and chicken hearts. Z. Zellforsch., 98t437-468. Sommer, J.R., and E.A. Johnson 1979 Ultrastructure of cardiac muscle. In: Handbook of Physiology. R.M. Berne, N. Sperelakis, and S.R. Geiger eds. Section 2: The Cardiovascular System, Volume I: The Heart. American Physiological Society, Bethesda, MD, pp. 113-186. Sommer, J.R., and R.A. Waugh 1976 The ultrastructure of the mammalian cardiac muscle cell-with special emphasis on the tubular membrane systems. A review. Am. J . Pathol., 82:192-232. Sperelakis, N., and R. Rubio 1971 An orderly lattice of axial tubules which interconnect adjacent transverse tubules in guinea-pig ventricular myocardium. J. Mol. Cell. Cardiol., 2211-220. 287 Tanaka, K., and T. Naguro 1981 High resolution scanning electron microscopy of cell organelles by a new specimen preparation method. Biomed. Res., [SupplI2:63-70. Tomita, Y., and V.J. Ferrans 1987 Morphological study of the transverse-axial tubular system (TAxTS) in rat heart using ferrocyanide-osmium method and thick sectioning. J . Submicrosc. Cytol., 19t523-535. Yoshikane, H., T. Nihei, and K. Moriyama 1986 Three-dimensional observation of intracellular membranous structures in dog heart muscle cells by scanning electron microscopy. J . Submicrosc. Cytol., 18:629-636.