Three dimensional arrangement of mitochondria and endoplasmic reticulum in the heart muscle fiber of the rat.код для вставкиСкачать
THE ANATOMICAL RECORD 200:139-151 (1981) Three Dimensional Arrangement of Mitochondria and Endoplasmic Reticulum in the Heart Muscle Fiber of the Rat D. SEGRETAIN, A. RAMBOURG, AND Y.CLERMONT Departement de bwbgie, Centre &Etudes N w h i r e s de Saclny, 91190 Gif-sur-Yvette, France ABSTRACT The three-dimensional arrangement of mitochondria and endoplasmic reticulum was studied in thick sections of the heart left ventricle fixed in glutaraldehyde and impregnated with the Ur-Pb-Cu technique and in thin sections of glutaraldehyde-fixed tissue post-fixed in potassium ferrocyanide-reduced osmium. Squarish flattened mitochondria, approximately the size of a sarcomere, were arranged in longitudinal columns in the clefts between the myofibrils. At the periphery of the fiber, the endoplasmic reticulum took the appearance of a subsarcolemmal network of plate-like and tubular cisternae running parallel to the cell surface. Between the myofibrils, the ER network formed longitudinally oriented repetitive units whose structure varied according to their position in relation to the A- or I- bands of the myofibrils. In front of the A-band, the endoplasmic reticulum appeared as a single layered network of anastomotic tubules compressed between the adjacent myofibrils. In front of the I-band, it formed a multilayered network the three-dimensional arrangement of which was dependent upon the presence or absence of the T-tubule. In the absence of the T-tubule, the ER cisternae were loosely anastomosed and occasionally displayed bulbous terminal swellings. In the presence of T-tubules, tubular ER cisternae were seen running parallel on both sides of the T-tubules and were continuous with sheetlike cisternae sandwiched between the distended T-tubule and adjacent extremities of longitudinally arranged mitochondria. These tubular or flattened cisternae were connected to each other by numerous bridging cisternae around the Ttubules. Since the pioneer study of Porter and Palade ('57) the ultrastructure of the cardiac muscle fiber has been investigated extensively (reviews by Fawcett, '61; James and Sherf, '68; Sommer and Jewett, '71; Simpson et al., '73; McNutt and Fawcett, '74; Sommer and Waugh, '76; Katz, '77). Three-dimensional models based on electron microscope observations of thin, sometimes serial, sections have been proposed by Porter and Palade ('57), Nelson and Benson ('63), Fawcett and McNutt ('69), Katz ('75), and Bossen et al. ('78). High voltage electron microscopy of thick sections combined with stereoscopic viewing has permitted a more direct appreciation of the three-dimentional configuration of selectively stained organelles. Thus, in cardiac muscle, the threedimensional organization of the T-system or the endoplasmic reticulum has been examined in the electron microscope after labeling the T-system with horseradish peroxidase and of the endoplasmic reticulum with lanthanum 0003-276)3/81/2002-0139$04.00 0 1981 ALAN R. LISS, INC. (Sommer and Waugh, '76). However, the low contrast obtained with such a microscope due to the acceleration of the electrons prevents the analysis of the fine three-dimensional details of the organelles. Staining techniques which impregnate mitochondria, cisternae of the endoplasmic reticulum, and elements of the Golgi apparatus have been utilized recently on thick sections to visualize with a n electron microscope a t 100 kV the three-dimensional architecture of the red muscle fiber in the rat diaphragm (Rambourg and Segretain, '80). It is the purpose of the present study to examine with the same staining methods the three-dimensional configuration of the rat heart muscle fiber with special emphasis on the structure of the endoplasmic reticulum and mitochondria as well as on the spatial relationship of these two organelles. Y. C.'s current addrees: Department of Anatomy, McGill University, Montreal, Quebec, Canada. Received August 13, 1980; accepted Oetober 27,1980. 140 D. SEGRETAIN, A. RAMBOURG, AND Y. CLERMONT MATERIALS AND METHODS Ten adult male rats were fixed by perfusion through the left ventricle with a 3.5% glutaraldehyde solution in 0.1M sodium cacodylate. After 15 minutes of perfusion, a portion of the left ventricle was excised and cut into small pieces that were immersed in the same fixative for an additional hour. They were then processed according to two different techniques. According to the first technique, the tissue was rinsed overnight a t 4°C in sodium cacodylate and then immersed for one hour a t room temperature in a 1:l mixture of 2% aqueous osmium tetroxide and 3% aqueous potassium ferrocyanide (Karnovsky, '71). This technique will be referred to as the reduced osmium technique. According to the second technique, the tissue was impregnated as described by Thi6ry and Rambourg ('76).After a quick rinse in distilled water, the tissue was immersed for one hour at 40°C in a 5% uranyl acetate aqueous solution, rinsed in distilled water, and post-stained for one hour at 40°C in a double lead and copper citrate solution prepared as indicated by Thi6ry and Rambourg ('76). Following several rinses in distilled water, the tissue was postfixed overnight a t 4°C in 1% unbuffered aqueous osmium tetroxide. This technique will be referred to as the Ur-Pb-Cu technique. Following these treatments, the tissue blocks were dehydrated in ethanol and embedded in Epon. Thin and thick (0.5-1 pm) sections were prepared with a MT1 Porter-Blum ultramicrotome, mounted on formvar coated grids and examined with the electron microscope. Thin sections of tissue postfixed in reduced osmium were counterstained for two minutes with lead citrate and examined at 60 kV in a Philips 301 (or 400) electron microscope. Thick sections of Ur-Pb-Cu impregnated material were examined without counterstaining at 80 or 100 kV. For stereoscopy, thick sections were placed on the goniometric stage of the electron microscope, and two photographs of the same field were taken after tilting the specimen a t -6" and +6" from the original 0" position of the goniometric stage. A three-dimensional view of the impregnated structure was obtained by looking a t properly adjusted pairs of such pictures with a stereoscopic binocular lens. RESULTS The mitochondria were well demonstrated with both staining techniques. In cross or slightly oblique sections (Figs. 3, 7, 121, the mitochondrial profiles located between the myofibrils varied considerably in size and shape. The longitudinal sections (Figs. 4, 5, 13), they formed longitudinal or slightly oblique columns. Individual members of these mitochondrial columns were frequently rectangular, flattened, and slightly curved elements. They were in register with a myofibrillar zone demarcated by two Z lines and were usually narrower at their equator in the region facing the H band (Figs. 10, 13). Their extremities frequently showed transverse expansions that ran parallel to each other on each side of the H band (Fig. 15). In the perinuclear cytoplasm spheroidal mitochondria accumulated in clusters. In thick sections of heart muscle fibers impregnated with Ur-Pb-Cu, the endoplasmic reticulum showed a repetitive pattern along the myofibrils (Fig. 5, 6). In register with the Abands, the cisternae, generally tubular in appearance, were mostly oriented parallel to the myofibrils and showed numerous short arched lateral connections (Fig. 2). In front of the H bands, these tubular cisternae formed a tight anastornotic network which did not show any definite orientation in relation to the long axis of the fiber (Figs. 2, 5, 6). In several places along this ER network, but mostly facing the junctions of the A and I bands, the tubular cisternae merged to form plate-like cisternae (Figs. 2, 6). In stereopairs of thick longitudinally or obliquely cut fibers, the ER network facing the A bands appeared as curtain-like structures with the cisternae aligned in a single plane (Figs. 12, 13). In reduced osmiumstained thin sections of longitudinally cut fibers, the overall organization of the ER network remained similar to the one previously described,although the cisternae were slightly more distended than in sections stained with Ur-Pb-Cu (Fig. 4). In cross sections of muscle fibers stained with reduced osmium, the linear rows of cisternal profiles, a t the level of the Abands, clearly indicated that the cisternae were distributed within a single plane a t the interface of myofibrillar territories (Fig. 7). The arrangement and distribution of ER cisternae around mitochondria was visualized, in Ur-Pb-Cu stained preparations, even though the mitochondria were heavily impregnated with the metallic precipitate (Figs. 5, 10, 12, 15). The ER network surrounding mitochondria a t the level of the A-band was formed of longitudinally running tubular cisternae MITOCHONDRIA AND ER IN HEART MUSCLE FIBERS 141 A \- I I- Fig. 1. Diagrammatic representation of the tridimensional organization of the endoplasmic reticulum as seen in a heart muscle fiber stained by the Ur-PbCu impregnation procedUre. A t the H-band level (H), middle of the A band (A), the ER takes the appearance of a tight network of tubules. From this network, cisternae of ER are roughly parallel to the long axis of the myofibrils and extend in both directions toward the I-band level where they merge in large meshes over the A-I junction. Sometimes thew longitudinal cisternae join together to give rise to plate-like or flattened cisternae. Over the mitochondria (M), the longitudinal cisternae are more widely dieperaed and a tight network is seen to face the depression of the mitochondria. At the I-band level (I), cistemae of ER form a 3-Dnetwork made up of large polygonal meshes a n a s t o m 4 in all directions of space. In the absence of the T-tubule, this meahwork simply extended across the I-band and showed some terminal bulbous swellings (S).In the preeence of the Ttubule (t), ER were parallel on one or both sides of it. Over or below the T-tubule, some #hod tubular or Aatbned bridges of ER ensure longitudinal continuity. In the space between the tipa of aajacent mitochondria and facing the I-band, in absence of the T-tubule (upper right), the ER form a loose three-dimensional network of tubules. In presence of the T-tubule (lower right), ER cisternae are arranged in a casing around the distended Ttubule (t) and are in continuity with the longitudinally and tranavemlly running ER cisternae. Fig. 2. Endoplasmie reticulum and T-tubule in a 0.5-pm thick longitudinal section of a cardiac muscle fiber impreg nated with the Ur-Pb-Cutechnique. At the I-band level, tubular cisternae (arrows)are running parallel on each side ofthe T-tubule (TI. They are interconnected by longitudinal tubular or plate-like elements (P)that bridge in front and behind the T-tubules and the ER network (ER)extending in front of two adjacent sarcomeres. A tight network of tubules is observed at the H-band level (H). In the bottom of the picture, the contours of an unreactive T-tubule are indicated by a broken line. 2: 2 band. X 117,000. Fig. 3 Thin croea section through a cardiac muscle fiber stained with ferrocyanide-reduced osmium. Mitochondria (M) and sheets of endoplasmic reticulum (ER)delimit myofibrillar areas of various sizes and shapes. (m0. A region of close contact between mitochondria and endoplasmic reticulum is indicated by two vertical arrows. N: nucleus. x 15,200. Fig. 4 Thin longitudinal section through a cardiac muscle fiber stained with ferrocyanide-reducedosmium. Mitochondria (M) approximately the size of a m o m e r e are arranged in longitudinal columns betweem the myofibrils. A t the 2 band level (74, the lumen of transversely oriented Ttubule (T)becomes wider in the region comprised between the tips of two adjacent mitochondria. At the H-band level, the endoplasmic reticulum appears aa a fenestrated sheet G: glycogen. x 33,000. (arr~ws). 144 D. SEGRETAIN. A. RAMBOURG, AND Y.CLERMONT which were more dispersed than those seen in the adjacent areas (Fig. 12). Facing the depression seen at the equator of the mitochondria which was in register with the H band, the tubular cisternae formed a tighter net. The ER network just described was usually seen along one or two surfaces of the mitochondria but frequently was also found to completely surround these organelles (Fig. 12). In thin sections stained with reduced osmium the tubular cisternae seen along the mitochondria were readily identified. They were occasionally found in longitudinal grazing sections of the mitochondria1 surface (Fig. 9) or appeared, in cross sections, as membranous profiles either isolated or in linear arrays closely associated with the surface of mitochondria (Figs. 3, 7). Continuous with the ER cisternae seen in register with the A-band, the cisternae facing the I-band formed a three-dimensional multilayered anastomotic network with large polygonal meshes (Fig. 2). This network was particularly evident in Ur-Pb-Cu stained thick sections viewed in stereopairs (Figs. 13, 14). In the absence of the T-tubule, this meshwork, formed of tubular or plate-like cisternae, simply extended across the I-band (Figs. 5, 6, 13). Some of these tubular cisternae terminated with bulbous swellings which were heavily impregnated with Ur-Pb-Cu (Figs. 5,6,13). Such terminal swellings were usually seen in small clusters and were connected to the lattice of anastomotic ER tubules (Figs. 6, 13). In the presence of the T-tubule, some ER cisternae formed tubular structures that ran parallel, on one or both sides, of the T-tubule (Figs. 2, 14). Such ER cisternae were connected to each other by short bridging tubular cisternae running above or below the T-tubule (Figs. 2,14). Other ER cisternae assumed a flattened and curved shape with their concave face applied to the surface of the T-tubule. Such incomplete sleeve-like structures continuous with the rest of the ER network were seen along the distended or non-distended segments of the T-tubule (Figs. 2, 14). In the space seen between the tips of adjacent mitochondria, which were facing the I bands, the ER cisternae formed, in the absence of the T-tubule, a loose three-dimensional network of anastomotic tubules. In presence of the T-tubule, which was usually distended in this location, the ER cisternae formed a casing around it made up of curved, flattened cisternae connected to each other by short anastomosing tubular cisternae (Fig. 15). The flattened cisternae were lodged in the narrow spaces located between the extremities of the mitochondria and the T-tubule (Fig. 15). This system of ER cisternae was continuous with the cisternae running longitudinally and parallel to the surface of mitochondria described above. In thin sections stained with reduced osmium, the multilayered arrangement of the ER cisternae facing the I bands along the myofibrils was readily detected under the form of superimposed layers of membranous profiles (Fig. 8). Similarly, the close association of flattened ER cisternae with the surface of the distended T-tubule located in the intermitochondrial spaces was clearly and frequently identified in such a material (Fig. 4). In the subsarcolemmal cytoplasm, flattened cisternae were interconnected by tubular elements (Fig. 11) and formed a well-developed network of endoplasmic reticulum running parallel to the cell surface. DISCUSSION Recent reviews on the ultrastructure of the heart muscle fiber (McNutt and Fawcett, '74; Katz, '77) have emphasized the pleomorphism of mitochondria which, in living cells, vary constantly in shape and distribution. In the present study, the examination of the threedimensional distribution of mitochondria has confirmed earlier observations (Moore and Fig. 5 Overall architecture of the endoplasmic reticulum as seen in 9.5-pm thick section of a cardiac muscle fiber impregnated with the Ur-Pb-Cu technique. The endoplasmic reticulum forms longitudinally oriented repetitive units the structure of which varies according to their position in front of the A or the I bands. In h n t of the Aband, numerous elongated flattened cisternae run parallel to the main axis of the fiber (horizontal arrows). Towards the mid portion of the A-band, they merge with a tight In network of tubules located in front of the H-band (H). front of the I-band and Z line (Z), the tubular cistemae are more loosely anastomosed and display bulbous Swellings (arrowhead). Plate-like cisternae, in the vicinity of the AI junction are labeled P. The strongly impregnated mitochondria (MI are arranged in longitudinal columns. x 35,000. Fig. 6. Endoplasmic reticulum as seen in a 0.5-pm thick longitudinal section of a cardiac muscle fiber. Ur-Pb-Cu technique. Fmm the tight network of tubules located in front of the H-band (H), elongated flattened cisternae run parallel to the long axis of the fiber towards the A-I junction where they are interconnected by numerous lateral arched connections. At the Z-band level (Z), in the middle part of the picture, the loosely anastomoaed cisternae are terminated by bulbous endings (arrows). In the lower part of the picture, a plate-like cisternae (P) is seen to bridge transversely oriented ER elements over an empty space that corresponds to an unstained T-tubule (T)the contours of which are indicated by a dotted line. Other plate-like cisternae (arrowheads) are seen at the A-I junctions. x 90,OOO. MITOCHONDRIA AND ER IN HEART MUSCLE FIBERS 145 Fig. 2. Endoplasmie reticulum and T-tubule in a 0.5-pm thick longitudinal section of a cardiac muscle fiber impreg nated with the Ur-Pb-Cutechnique. At the I-band level, tubular cisternae (arrows)are running parallel on each side ofthe T-tubule (TI. They are interconnected by longitudinal tubular or plate-like elements (P)that bridge in front and behind the T-tubules and the ER network (ER)extending in front of two adjacent sarcomeres. A tight network of tubules is observed at the H-band level (H). In the bottom of the picture, the contours of an unreactive T-tubule are indicated by a broken line. 2: 2 band. X 117,000. Fig. 9. Thin longitudinal section through a cardiac muscle fiber stained with fermcyanide-reducedosmium. At the H-band level (H), a transversely oriented membranous profile (ER) is seen to leave at right angles to a longitudinally oriented cisternae of the endoplasmic reticulum (arrowheads). G glycogen; M: mitochondrion. x 105,000. Fig. 10. Thick (1 km) longitudinal section through a cardiac muscle fiber impregnated with the Ur-Pb-Cu technique. Longitudinally oriented ER cisternae (ER) are seen on each side of the squarish mitochondria (M)at the A-band level (A). In the upper part of the picture, plate-like cisternae (P) are sandwiched between the distended T-tubdm (T)and the tips of the mitochondria. X 95,000. Fig. 11. Thick section (0.5 pm) parallel to the surface of a cardiac muscle fiber impregnated with the Ur-Pb-Cu technique. The subsarcolemmal network of endoplasmic r e ticulum is made up of platelike cisternae (arrows) interconnected by tubular elements. M: mitochondria; mf myofibril. x 33,000. 148 D. SEGRETAIN, A. RAMBOURG, AND Y.CLERMONT Figures 12-15 are stereopairs. The two membera of each pair of photographs of the same field are taken at different angles ( f7")h m the original 0" position of the goniometric stage. A single stereoscopic image of the impregnated organelles may be obtained by using a stereoscopic binocular lens, the distance between the central axes of the two lenses being adjusted to 65-70 mm. All figures were taken from 1-pm thick sections of cardiac muscle fibers impregnated by the Ur-Pb-Cu technique. Fig. 12. Overall distribution of endoplasmic reticulum and mitochondria as seen in an oblique section. The endoplasmic reticulum at the A-band level (A) forma a single sheet of densely packed elements which is more loosely anaatomosed and displays bulbous terminal endings at the Iband level (I). Note the close relationship between mitochondria (M)and endoplasmic reticulum (arrowhead). x 11.700. Fig. 13. Longitudinal section showing the difference in the three-dimensional arrangement of the endoplasmic reticulum at the I- and A-band levels. Whereas the H-band network and the longitudinal cisternae at the A-band level are mainly located in a single plane, the tubular cisternae at the I-band level are oriented and anastomosed in all directions of space (arrows). Bulbous terminal swelling are also seen in this location. Between adjacent extremities of two longitudinally arranged mitochondria (MIan unstained T-tubule (TI is surrounded by heavily stained flattened cistemae. x 25,000. Fig. 14. Three-dimensional distribution of the endoplaamic reticulum and T-tubule at the I-band level. Transversely oriented tubular cisternae run on each side of the T-tubule (T) located over one half of the I-band on the left of a Z line (2). They are interconnected by small bridges (arrow) over and below the T-tubule. A flattened and slightly curved cistema may be seen in the lower half of the field (arrowhead). x 43,000. Fig. 15. Three-dimensionalarrangement of endoplasmic reticulum and mitochondria at the T-tubule level. In this longitudinal thick section, the mitochondria (M)give rise to short transverse expansions which are surrounded by a network of tubular ER cisternae. At left, the two flattened ER cisternae running parallel to the transverse expansions of two adjacent mitochondria (arrows) are interconnected by a loose network of tubular elements making up a caselike structure around the unstained T-tubule. x 19,500. MITOCHONDRIA AND ER IN HEART MUSCLE FIBERS 149 150 D. SEGRETAIN, A. RAMBOURG, AND Y. CLERMONT Ruska, '57; Porter and Palade, '57; Muir, '67; Fawcett and McNutt, '69) and shown that squarish flattened mitochondria, approximately the size of a sarcomere, are arranged in longitudinal columns in clefts between the myofibrils. Earlier studies on the structural organization of the endoplasmic reticulum in cardiac muscle fiber have demonstrated clearly the transverse and longitudinal continuity of this intracytoplasmic membranous system throughout the muscle fibers (Porter and Palade, '57; Fawcett and McNutt, '69). This morphological feature was amply verified in the present study on thick sections stained with Ur-Pb-Cu. Furthermore, the variability of its configuration in various animal species was stressed (McNutt and Fawcett, '74; Sommer and Waugh, '76). Thus, for example, the existance of a network of tubular cisternae, in front of the H band, described in the heart muscle of hamster and mouse (Sommer and Waugh, '76) and in the rat, as shown in the present study, was not observed in the cat myocardium (Fawcett nd McNutt, '69). These various studies have not, however, stressed the differences in the three-dimensional organization of the ER cisternae at the level of the A- and I-bands or presented detailed descriptions of the three-dimensional relationships of the ER cisternae with the T-tubules or mitochondria. When stereopairs of thick sections stained with Ur-Pb-Cu are examined, the ER a t the A-band level appears as a single sheet of anastomotic tubular or plate-like cisternae, whereas a t the I-band level it forms a multilayered network (Fig. 1).In the absence of Ttubules this loose network of ER tubular cisternae in front of the Z line is often associated with clusters of bulbous terminal swellings (Fig. 1).These terminal bulbs seemingly correspond to the coated vesicles observed in thin sections by Fawcett and McNutt ('69) and thought to bud or fuse with the endoplasmic reticulum. Sommer and Waugh ('76) working with the high voltage EM on 1-pm thick sections observed, as in the present investigation, that such membranous profiles were always continuous with the endoplasmic reticulum. The functional role of these clusters of terminal ER swellings remains to be discovered. In the presence of T-tubules the ER cisternae were seen to assume a variable configuration. Frequently, however, tubular cisternae, continuous with the rest of the ER network, were found to run transversely, parallel, and on each side of the T-tubules. Simpson ('65) and Simpson and Rayns ('68) have emphasized the existence of such transverse tubules following the Z line and called them "Z-tubules." McNutt and Fawcett ('74) indicated that such tubules did not differ from ER cisternae. The present description is in accord with this view and further demonstrates the existence of numerous short tubules bridging these transversal tubules over and below the interposed T-tubule (Fig. 1). In models prepared from thin sections (Perter and Palade, '57; Nelson and Benson, '63; Fawcett and McNutt, '69; Katz, '75) areas of close contact between ER and the T-tubule were usually shown as blind-end, foot-like expansions of the cisternae applied to the surface of the T-tubule. In Ur-Pb-Cu stained thick sections such foot-like ER cisternae were not seen. While the T-tubule-associated ER cisternae assumed a flattened and curved shape closely apposed to the surface of the T-tubule, as demonstrated by Simpson and Rayns ('68), Farbes et al. ('771, and Forbes and Sperelakis ('771, they were shown in the present study to be continuous by means of numerous branches with the rest of the ER network (Fig. 1).Furthermore, the analysis of thick sectons also revealed that zones of close contact between endoplasmic reticulum and distended segments of the T-tubule were found preferentially in the sarcoplasm located between the extremities of mitochondria and the interposed T-tubule (Fig. 1).The functional significance of this association: T-tubules-ER cisternae-mitochondria remains to be clarified, Rambourg and Segretain ('80) in their threedimensional study of the ER of red muscle fibers of the rat diaphragm speculated that the bidimensional and three-dimensional configuration of the ER network facing the A- and I-bands, respectively, represented an adaption of this organelle t o the selective shortening of the myofibrils at the I-band level during contraction. In the cardiac muscle fibers some of the tubular ER cisternae were shown to be attached to the Z-line by a cytoplasmic filamentous material (Simpson and Rayns, '68; Edge and Walker, '70; Jewett et al., '71). During contraction the ER cisternae would be stabilized at that point and be prevented from moving out of the I bands, but, simultaneously, would form a t this level a compact multilayered network. Furthermore, since the mitochondria are likely to be maintained in position by the endoplasmic reticulum, the MITOCHONDRIA AND ER IN HEART MUSCLE FIBERS relationship between mitochondria, endoplasmic reticulum, and T-tubule would remain constant during the cycle of contraction and relaxation. ACKNOWLEDGMENTS This work was supported by a France-Quebec Exchange program. The work done a t McGill University was supported by the Medical Research Council of Canada. The help of Dr.L. 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