Three-dimensional architecture of sarcoplasmic reticulum and T-system in human skeletal muscle.код для вставкиСкачать
THE ANATOMICAL RECORD 218~275-283(1987) Three-Dimensional Architecture of Sarcoplasmic Reticulum and T-System in Human Skeletal Muscle KAZUKO HAYASHI, RODMAN G. MILLER, AND A. KEITH W. BROWNELL Department ofAnatomy (K.H., R. G.M.) and Department of Clinical Neurosciences (A.K.W.B.), Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4 N l ABSTRACT A modified Golgi method combined with stereoscopy has been used to demonstrate the three-dimensional architecture of the sarcoplasmic reticulum (SRI and the T-system in human skeletal muscle. SR formed a continuous repeating network with a different structure dependent upon the sarcomere position. Intermyofibrillar SR contained three regions: 1)fenestrated collars overlying the M-band region, 2) terminal cisternae overlying the A-I region, and 3) a three-dimensional anastomosed tubular network overlying the Z-band region. Longitudinal andlor transverse SR tubules connected these regions. Subsarcolemmal SR was also composed of three regions: 1)transversely oriented polygonal meshes overlying the Mband, 2) single-layered tubules overlying the Z-band region, and 3) a loose network between the two. In the subsarcolemmal sarcoplasm, where mitochondria were aggregated, SR anastomosed loosely and showed nonfenestrated cisternae beneath the plasma membrane. The T-system was composed of transversely oriented networks overlying the A-I region with occasional longitudinal tubules connecting these networks. Extensive ultrastructural studies of human skeletal muscle have been done, with thin sections, which describe the two-dimensional appearance of different fiber types and pathological conditions. Less is known about the three-dimensional architecture of the sarcoplasmic reticulum (SRI and T-system in normal human muscle, undoubtedly due to the sparsity of en face visualizations of the SR and T-system in thin sections. Selective SR staining techniques utilizing high-voltage electron microscopy have recently been developed to study thick sections of frog muscle (Bailey and Peachey, 1975), and various staining methods using conventional electron microscopy have been successfully applied to obtain three-dimensional visualizations of the intermyofibrillar membrane systems in animal skeletal muscle (Forbes and Sperelakis, 1980; Rambourg and Segretain, 1980; Scales and Yasumura, 1982; Yasumura and Scales, 1982). In the present communication, we have used the modified Golgi black reaction (the Golgi method) initially applied to skeletal muscle by Veratti (1902) and modified for electron microscopy by Franzini-Armstrong and Peachey (1982) to study the morphology of the SR and the T-system in human skeletal muscle. Although tissue treated by this method does not stain homogeneously, adequate staining could be obtained to allow us to better define the three-dimensional morphology of the SR and the T-system in normal human skeletal muscle. MATERIALS AND METHODS The vastus lateralis biopsy specimens used in this study were obtained from 31 subjects (ten males and 21 females, aged from 4 to 64 years, mean age 32 years) who were undergoing testing for malignant hyperther0 1987 ALAN R. LISS, INC. mia susceptibility but who turned out to be normal. In addition, histological and histochemical examination of these muscles also showed no abnormality.’ Muscle bundles, 20 mm in length and 2-3 mm in diameter, were slightly stretched, tied to a wooden stick, and fixed immediately in 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 at 4°C for 1 day to 2 weeks. For the Golgi method, the bundles were cut into pieces (2 x 2 x 1 mm), rinsed three times for 20 min each in 0.1 M cacodylate buffer, washed twice for 30 min each in 3% K2Cr207, and incubated in a solution containing 0.2% Os04, 2.4% K2Cr207 for 8 days a t 4°C and then in 0.75% AgN03 a t 4°C for 5 days. Second infiltrations in the same solutions (0.2% Os04 and 2.4% K2Cr207 and 0.75% AgN03) followed, with 3 and 2 days of incubation, respectively. Specimens were then dehydrated in ethanol, infiltrated with propylene oxide, and embedded in Spurr’s plastic media (Spurr, 1969). Thick sections (0.3-1.0 pm) were cut from the blocks treated by the Golgi method and scanned light microscopically for adequately staining regions. The blocks were trimmed to these regions and cut for EM observation. Sections were mounted on folding oyster grids which were pressed firmly together and without further staining were examined in a Philips 400 electron microscope at 100 kV. Stereo pairs of electron micrographs of Received April 24, 1986; accepted January 27, 1987. Address reprint requests to Dr. A.K.W. Brownell, Department of Clinical Neurosciences, Faculty of Medicine, University of Calgary, Foothills Hospital, 1403 29th Street NW, Calgary, Alberta, Canada T2N 2T9. ‘These examinations were done in the Department of Pathology at the Foothills Hospital in Calgary. 276 K. HAYASHI, R.G. MILLER, AND A.K.W. BROWNELL the same field were made by tilting the specimen stage a t +6-12" and viewed with a stereoscope. For comparing SR morphology between fiber types, we prepared serial thin and thick sections from specimens treated by the Golgi method and identified fiber types on the basis of the Z-band width and M-band appearance (Sjostrom et al., 1982) in thin sections which were additionally stained with uranyl acetate and lead citrate (Reynolds, 1963). The structure of the SR was then examined in the serial thick section. We also used a series of thin and thick sections to confirm the identity of the structures stained with the Golgi method. Additional tissue was processed for conventional electron microscopy for examination of SR and T-tubule morphology. Strips of the muscle fixed in glutaraldehyde were postfixed in 1% OsO4 in 0.1 M cacodylate buffer, pH 7.4, a t 4°C for 1 hr, dehydrated in ethanol, and embedded in Spurr's plastic media (1969). Th'in sections were cut on a n ultramicrotome and stained with uranyl acetate and lead citrate (Reynolds, 1963). RESULTS In our Golgi preparations of human skeletal muscle, both the SR and the T-system were frequently stained, while in some regions only the T-system or the SR was stained, and in other regions the staining was totally lacking (Fig. 1). Occasionally, nuclear membranes and mitochondria were also stained. In our comparative study of fiber types, although morphometric differences have been reported by Eisenberg (1983), we could not find any predictable difference in the morphology of SR (Fig. 1). We found variability of SR complexity between subjects rather than between fiber types; however, no quantitation of this variability was attempted. Therefore, we shall make no further distinction of structures on the basis of fiber types. Sarcoplasmic Reticulum Sarcoplasmic reticulum (SR) in human skeletal muscle showed a different structure depending upon its relation both to the plasma membrane and to the adjacent myofibrillar pattern; we have termed these the subsarcolemmal SR and the intermyofibrillar SR, respectively. Between myofibrils, the SR was composed of longitudinally oriented repeating units whose morphology varied according to its position within the sarcomere (Figs. 1, 2). Overlying the M-band region, SR formed patchlike fenestrated collars of varying size and shape (Fig. 2). Although these collars were usually transversely connected by thinner tubules, they were not always continuous around myofibrils (Fig. 2). Overlying the A-I junction, SR associated with the Ttubule formed distended sacs, i.e., terminal cisternae (Figs. 1, 2). These terminal cisternae were apposed to the T-tubule, forming triads, occasionally dyads, and rarely tetrads or pentads. These sacs of various sizes were usually located perpendicular but also occasionally parallel to the fiber axis. Adjacent terminal cisternae were connected by one or two thinner transverse tubules running parallel to but separate from the T-tubule (Figs. 1, 2), and the two terminal cisternae making a triad were usually connected by small, longitudinally oriented tubules located at the lateral region of the triads (Fig. 2b, double arrowheads). Longitudinal tubules provided connection between SR in the M-band region and in the A-I junction region. SR overlying A-bands formed a single layer between adjacent myofibrils, while SR overlying I-bands had a more complex multilayered structure (Figs. 1,2). In the Z-band region, tubular forms of SR irregularly anastomosed to form a three-dimensional network, encircling each myofibril and displaying horizontal continuity (Figs. 1, 2). The SR of this region also had occasional fenestrations. The SR between the Z-band and the A-I junction was connected by longitudinal SR tubules (Figs. 1,2). At the periphery of the fiber, the SR had a completely different appearance (Figs. 3-5). Here, it was composed of transversely oriented continuous polygonal meshes at the M-band region, transverse tubules at the Z-band region, and a loose network of SR connecting the two (Fig. 3). These subsarcolemmal regions were continuous with the intermyofibrillar regions (Fig. 3). Some parts of the network were distended to form nonfenestrated cisternae which did not have any special association with T-tubules, as was seen in the deeper portions of SR. Other parts occasionally made triads with T-tubules. Since these findings could only be clearly resolved in fortuitous tangential thick sections in which the SR was stained without extracellular staining (Figs. 3, 51, we used the goniometer specimen stage to confirm these findings in thick sections where plasma membranes were not tangentially sectioned. We occasionally observed the subsarcolemmal network in tangential thin sections prepared by conventional methods (Fig. 4a), thus confirming our Golgi method observations. In the subsarcolemmal mitochondria-rich region, tubular and occasionally focally bulged SR loosely anastomosed in all directions among other organelles (Fig. 5). Beneath the plasma membrane, in addition to a tubular appearance, the SR in this area also formed small, nonfenestrated cisternae without any specific orientation (Fig. 5).T-tubules were usually not observed in this area in Golgi preparations (not shown). Although junctional feet located between a subsarcolemma1 vesicle and plasma membrane were not observed in Golgi preparations, they were occasionally seen in conventionally prepared thin sections (Fig. 4b). Therefore, nonfenestrated cisternae located beneath the plasma membrane would form a peripheral coupling with plasma membrane in human as in other skeletal muscles (Forbes et al., 1977; Forbes and Sperelakis, 1980; Spray et al., 1974). T-System In longitudinal sections stained by the Golgi method, T-tubules were easily distinguished from SR; the T-tubules were much more electron dense (Figs. 1, 2) and were located close to the A-I junction (Fig. 6a). We occasionally observed longitudinal T-tubule connecting two transversely oriented T-tubule networks and sometimes being part of a longitudinally oriented triad (Fig. 6a). These longitudinally oriented T-tubules were not evenly distributed throughout the fiber, but they were common in regions of dislocation of cross-striations or in the regions close to the plasma membrane. In cross sections, T-tubules showed both flat ribbon and cylindrical shapes and formed a continuous transverse network (Fig. 6b). Although the flat ribbon T-tubules typically associated with the SR in the formation of triads were SARCOPLASMIC RETICULUM IN HUMAN MUSCLE Fig. 1. Electron micrograph of a thick section (0.5 pm in thickness) prepared by the Golgi method includes three fibers; both left (A) and right (C) fibers are type I1 and the middle one (B)is type I. The staining is observed on sarcoplasmic reticulum (SR) in fiber A, SR and some mitochondria in fiber B, both SR and T-tubule in fiber C, and extracellular spaces (EC) between fibers. SR shows repeating units depending on myofibrillar pattern. Bar = 1 pm. 277 Fig. 2. Stereo pair (Za)and a higher magnification (2b) of the same section in which SR and T-tubules are selectively stained. Intermyofibrillar SR shows a continuous network made up of repeating units: patchlike fenestrated collars overlying the M-band (FC), terminal cisternae overlying the A-I region forming triads (arrows) with T-tubule, and three-dimensionally anastomosed tubules overlying the Z-band region. Longitudinal andor transverse tubules connect these regions Figure 2b (arrowheads). Double arrowheads point to the small tubules that connect the two terminal cisternae of a triad. Bar = 1 pm. Tilt 12". + 278 K. HAYASHI, R.G. MILLER, AND A.K.W. BROWNELL Fig. 3. a: Slightly oblique section (0.5 pm in thickness) prepared by the Golgi method in which only SR is stained showing the structure of the SR in the periphery of a fiber. The elaborate network of subsarcolemma1 SR can be easily observed in the upper region of the centre fiber where there is no coarse precipitation in the extracellular space (EC). Bar = 1 pm. b: Stereo pairs of the same section as above. The three-dimensional architecture of the subsarcolemmal SR and conti- nuity with the intermyofibrillar SR can be detected. Bar = 1 pm. Tilt 6". c: A higher magnification of the same section as 3b shows the three regions of the sarcolemmal SR transversely oriented polygonal mesh (arrows) overlying the M-band region, single-layered tubules overlying the Z-band region (double arrowheads), and loose networks between the former two which contain nonfenestrated cisternae (arrowheads). Bar = 0.5 pm. SARCOPLASMIC RETICULUM IN HUMAN MUSCLE Fig. 4. a: A tangential section prepared by conventional methods and stained with uranyl acetate and lead citrate shows a region of the subsarcolemmal SR network polygonal meshes overlying the M-band region (arrow) and nonfenestrated cisternae (arrowheads). Double arrowheads indicate caveolae. EC: extracellular space. Z: Z-band. Bar = 279 0.1 pm. b: A longitudinal section prepared by conventional methods and stained with uranyl acetate and lead citrate shows junctional feet (arrowheads) between plasma membrane (Pm) and subsarcolemmal cisternae. Z: Z-band. C: caveolae. EC: extracellular space. Bar = 0.1 pm. Fig. 5. Stereo pair in which only SR is stained. This oblique section shows a loose network of SR in the subsarcolemmal region where mitochondria (M) and lipid droplets are aggregated. Beneath the plasma membrane, SR contains nonfenestrated cisternae (arrowheads) in the tubular anastomosed network. Arrows indicate bulged SR. Intermyofibrillar network of SR is seen in the upper left region and polygonal meshes in the subsarcolemmal region are seen in the upper right region. Bar = 1pm. Tilt f 6". a) 280 K. HAYASHI, R.G. MILLER, AND A.K.W. BROWNELL Fig. 6. a: Longitudinal stereo pair in which only T-tubules are Bar = 1 pm. Tilt k 12". b Transverse stereo pair in which only Tstained, showing T-tubule networks located at the A-I region and tubules are stained, showing a continuous transverse network of Toccasional longitudinal tubules (arrowheads). Arrow shows flat, rib- tubules forming both flat ribbon and cylindrical shapes. Bar = 1 pm. bon-shaped regions of T-tubules forming longitudinally oriented triads. Tilt k 12". the commonest type of T-tubule, they were less frequent than was seen in frog twitch muscle or rat white muscle (Peachey and Franzini-Armstrong, 1983). In the silver-stained preparations we could follow the course of T-tubules close to the plasma membrane; however, we were unable to define connections between the T-tubule and the plasma membrane due to the presence of artifactual silver precipitate frequently seen in the extracellular space and sometimes in the intracellular space. Evidence of openings of the T-tubules to the extracellular space were only rarely seen in the conventionally prepared thin sections (Shafiqet al., 1966);T-tubules SARCOPLASMIC RETICULUM IN HUMAN MUSCLE 281 d Fig. 7. A diagrammatic presentation of a superficial portion of a human skeletal myofiber demonstrating the structural characteristics of subsarcolemmal and intermyofibrillar membrane systems. 1:plasma membrane; 2: basal lamina; 3: endomysial collagen; 4: myofibril; 5: terminal cisternae; 6: fenestrated collar; 7: continuous tubular anasto- moses overlying the Z-band region; 8: longitudinal and/or transverse tubules; 9: T-tubule; 10: subsarcolemmal polygonal mesh overlying the M-band region; 11:single-layered tubules overlying the Z-band region; 12: a nonfenestrated cisternae; 13: mitochondria; A:A-band; 1:I-band; MM-band Z:Z-band. followed a sinuous course beneath the plasma membrane and appeared to open in the region where subsarcolemmal caveolae were found (Oguchi and Tsukagoshi, 1980). The disposition of the openings to the extracellular space is similar to that seen in other vertebrates (Rayns et al., 1968; Zampighi et al., 1975). Our findings are summarized and illustrated in Figure 7. conventionally prepared thin sections. The complex geometry of the SR, dependent on its relation to the plasma membrane and different parts of the contractile apparatus, has been demonstrated. These results are summarized and illustrated in Figure 7. Our investigations indicate that SR and T-tubules in human skeletal muscle are structurally similar to what has already been described in other mammalian muscles (Landon, 1982). The intermyofibrillar SR in human skeletal muscle shows three regions with respect to the adjacent myofibril pattern: 1)fenestrated collars overlying the M-band region, 2) terminal cisternae at the A-I region, and 3) three-dimensionally anastomosed tubular SR in the Z-band region. Longitudinally and/or transverse SR tubules connect these regions. We also fre- DISCUSSION We report the first successful application of the Golgi method in the study of human skeletal muscle to demonstrate the three-dimensional architecture of the SR and the T-system. Thick sections from the Golgi method demonstrated good structural preservation and detail of the SR and T-system that could not be appreciated in 282 K. HAYASHI. R.G. MILLER, AND A.K.W. BROWNELL quently found short longitudinal connections between ACKNOWLEDGMENTS opposite terminal cisternae forming a triad, a s has been reported in earlier studies (Peachey, 1965; Luff and AtSupported by the Alberta Heritage Foundation for wood, 1971; Franzini-Armstrong, 1973; Rambourg and Medical Research and the Muscular Dystrophy AssociaSegretain, 19801, though rarely demonstrated in conven- tion of Canada. tional thin sections of human skeletal muscle. Overall LITERATURE CITED our findings indicate that the SR in human skeletal muscle forms a continuous network made up of repeat- Bailey, C.H., and L.D. Peachey (1975) The sarcoplasmic reticulum of frog slow and twitch muscle fibers as revealed by stereoscopic high ing units throughout the fiber length, in agreement voltage electron microscopy. In: Annu. Proc. Electron. Microsc. Soc. with a n earlier study (Revel, 1962). Am., 33rd. C.H. Bailey, ed. Claitor’s, Baton Rouge, Louisiana, pp. Although the overall morphology of intermyofibrillar 552-553. SR in human skeletal muscle is generally similar to Eisenberg, B.R. (1983)Quantitative ultrastructure of mammalian skeletal muscle. In: Handbook of Physiology, Section 10, Skeletal Musthat in mammalian muscle, we noted, for example, 1) cle. L.D. Peachey, R.H. Adrian, and S.R. Geiger, eds. Am. Physiol. that the fenestrations at the A-I region described in the Soc., Bethesda, MD, pp. 73-112. mouse diaphragm muscle (Waugh et al., 1973) were not Forbes, M.S., B.A. Plantholt, and N. Sperelakis (1977) Cytochemical observed in human skeletal muscle and 2) that in rat staining procedures selective for sarcotubular systems of muscle: diaphragm muscle, the SR overlying the M-band region Modifications and applications. J. Ultrastruct. Res., 6Ot306-327. showed tubular anastomoses containing few fenestra- Forbes, M.S., and N. Sperelakis (1980) Membrane systems in skeletal muscle of the lizard Anolis carolinensis. J. Ultrastruct. Res., 73:245tions (Rambourg and Segretain, 1980) while in human 261. muscle and rabbit skeletal muscle (Yasumura and Franzini-Armstrong, C. (1973) Membranous systems in muscle fibers. In: The Structure and Function of Muscle. Vol. 2. G.H. Bourne, ed. Scales, 1981) SR contains fenestrated collars overlying Academic Press, New York and London, pp. 532-619. the M-band region. Furthermore, in our preparations, Franzini-Armstrong, C., and L.D. Peachey (1982) A modified Golgi although the SR was generally similar between subblack reaction method for light and electron microscope. J. Histojects, the degree of structural complexity of the SR chem. Cytochem., 3Ot99-105. Jorgensen, A.O., A.C.-Y. Shen, K.P. Campbell, and D.H. MacLennan varied. (1983) Ultrastructural localization of calsequestrin in rat skeletal The elaborate network of subsarcolemmal SR found muscle by immunoferritin labeling of ultrathin frozen sections. J. during this study has not been emphasized in previous Cell Biol., 97,1573-1581. reports. In conventionally prepared thin sections, the Kelly, A.M. (1971) Sarcoplasmic reticulum and T-tubules in differentiating rat skeletal muscle. J. Cell Biol., 49t335-344. subsarcolemmal SR was only described as subsarcolemD.N. (1982) Skeletal muscle normal morphology, development mal vesicles (Porter and Palade, 1957) and subsarcolem- Landon, and innervation. In: Skeletal Muscle Pathology. F.L. Mastaglia and mal cisternae (Schiaffino and Margreth, 1969; Kelly, J. Walton, eds. Churchill Livingstone, Edinburgh, pp. 1-87. 1971; Spray et al., 1974). The few previous reports Luff, A.R., and H.L. Atwood (1971) Changes in the sarcoplasmic reticulum and transverse tubular system of fast and slow skeletal (Forbes et al., 1977; Rambourg and Segretain, 1980; muscles of the mouse during postnatal development. J. Cell Biol., Forbes and Sperelakis, 1980) from thick sections com51t369-383. bined with selective staining methods only partially de- Oguchi, K., and H. Tsukagoshi (1980) An electron-microscopic study of scribed its architecture. Our study has revealed the the T-system in progressive muscular dystrophy (Duchenne) using lanthanum. J. Neurol. Sci., 44t161-168. complex structure of the subsarcolemmal SR in human L.D. (1965) The sarcoplasmic reticulum and transverse tuskeletal muscle. Its configuration depended upon its re- Peachey, bules of the frog’s sartorius. J. Cell Biol., 25:209-231. lationship to not only the sarcomere pattern but also to Peachey, L.D., and C. Franzini-Armstrong (1983) Structure and funcmitochondria-rich regions. tion of membrane systems of skeletal muscle cells. In: Handbook of Physiology, Section 10, Skeletal Muscle. L.D. Peachey, R.H. AdOur observation of subsarcolemmal cisternae suggests rian, and S.R. Geiger, eds. Am. Physiol. Soc., Bethesda, MD, pp. that peripheral coupling is a common form of junctional 23-71. complex in the subsarcolemmal region in human skele- Porter, K.R., and G.E. Palade (1957) Studies on the endoplasmic retictal muscle, as has been noted in adult skeletal muscle ulum. 111. Its form and distribution in striated muscle cells. J. Biophys. Biochem. Cytol., 3:269-300. from higher vertebrates (Spray et al., 1974). In addition, Rambourg, A., and D. Segretain (1980) Three-dimensional electron peripheral coupling has been repeatedly observed in demicroscopy of mitochondria and endoplasmic reticulum in the red veloping mammalian skeletal muscle (Kelly, 1971; muscle fiber of the rat diaphragm. Anat. Rec., 197:33-48. Schiaffino and Margreth, 1969; Walker et al., 19751, Rayns, D.G., F.O. Simpson, and W.S. Bertaud (1968) Surface features of striated muscle 11. Guinea pig skeletal muscle. J. Cell Sci., where the mature T-tubule network has not yet devel3:475-482. oped. J.P. (1962) The sarcoplasmic reticulum of the rat cricothyroid Functional correlations might be expected to exist that Revel, muscle. J. Cell Biol., 12571-588. would correspond to the variable anatomy. It has been Reynolds, E.S. (1963) The use of lead citrate at high pH as a n electronopaque stain in electron microscopy. J. Cell Biol., 17t.208-212. suggested that the variable structure of the intermyofibrillar SR is a n adaptation to the shortening and length- Scales, D.J., and T. Yasumura (1982) I. Stereoscopic views of a dystrophic sarcotubular system: Selective enhancement by a modified ening reaction of sarcomeres (Revel, 1962). Also, funcGolgi stain. J. Ultrastruct. Res., 78:193-205. tional correlation has been noted for the terminal cister- Schiaffno, S., and A. Margreth (1969) Coordinated development of the sarcoplasmic reticulum and T-system during postnatal differentianae (Jorgensen et al., 1983). Further studies will be tion of rat skeletal muscle. J. Cell Biol., 41:855-875. required to determine whether a s rich a degree of func- Shafiq, S.A., M. Gorycki, L. Goldstone, and A.T. Milhorat (1966) Fine tional specialization will be demonstrated as has been structure of fiber types in normal human muscle. Anat. Rec., demonstrated anatomically in the SR. 156:283-302. 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