Cell Tiss. Res. 157, 1--16 (1975) 9 by Springer-Verlag 1975
Distribution and Dimension of the T-System in Different Muscle Fiber Types in the Atlantic Hagfish (Myxine glutinosa, L.) H e l g e K o r n e l i u s s e n a n d K ~ r e Nicolaysen* Anatomical Institute and Institute of Neurophysiology, University of 0slo, 0slo, Norway Received December 16, 1974
Summary. Triad density relative to sarcomeres, size of T-system tubules, sarcomere length, muscle fiber diameter in native and fixed states, and size of myofibrils were measured in four striated muscle fiber types in Atlantic hagfishes (Myxine glutinosa, L.) of different sizes. Triads occur at A/I-junctions in all fiber types. The density of triads relative to sarcomeres is higher in "white" than in " r e d " muscle fibers. The T-tubules show no sign of branching. The area of the T-system tubules is 3 4 times the surface area in 80 ~m "white" muscle fibers and 1-2 times that in 60 ~m " r e d " fibers. The size of myofibrils is similar in "white", "intermediate", and " r e d " fibers of m. parietalis, and constant through a large span of animal size. In "white" fibers, increase in diameter up to 90 ~zm is accompanied by an increase in the number of myofibrils, not by an increase in the individual size of the myofibrils. Above 90 ~m," white" fibers grow by increasing the amount of intermyofibrillar space. This is reflected by an extensive shrinkage of the thicker "white" fibers during the preparative procedure for electron microscopy, a shrinkage that is limited only by complete packing of the myofibrils. " R e d " fibers shrink much less. Key words: Muscle fiber types (Myxine glutinosa, L.) - - T - s y s t e m - G r o w t h - Shrinkage - - Electron microscopy. Introduction I n a previous p u b l i c a t i o n we defined four u l t r a s t r u c t u r a l l y different muscle fiber t y p e s in t h e A t l a n t i c hagfish (Myxine glutinosa, L.) (Korneliussen a n d Nicolaysen, 1973). R e c e n t i n v e s t i g a t i o n s b y one of us (Nicolaysen, 1974 a, b) p r o m p t e d a closer analysis of t h e T - s y s t e m in this animal. As r e v i e w e d b y S m i t h (1966) a n d F r a n c i n i - A r m s t r o n g (1973) t h e t r i a d s m e d i a t e t h e e x c i t a t i o n - c o n t r a c t i o n coupling in skeletal muscle fibers. T h e Ts y s t e m t u b u l e s (Andersson-Cedergren, 1959) form t h e central e l e m e n t s of t h e t r i a d s . T h e y are open a t t h e surface, a t least t o some e x t e n t , a n d t h u s t h e s y s t e m r e p r e s e n t s a g e o m e t r i c a l l y c o m p l i c a t e d e x t e n s i o n of t h e e x t r a c e l l u l a r space. T h e c o n t i n u i t y w i t h t h e e x t r a c e l l u l a r space has been d e m o n s t r a t e d b y t h e e n t r a n c e of e x t r a c e l l u l a r t r a c e r s into t h e tubules. T h e surface d e p o l a r i z a t i o n resulting from a c t i v i t y in t h e m o t o r axons is c o n d u c t e d i n w a r d s along t h e T - t u b u l e s . S o m e h o w
Send o]/print requests to: Dr. H. Korneliussen, Anatomical Institute, University of Oslo, Karl Johansgt. 47, Oslo 1, Norway. * We thank Dr. J. K. S. Jansen, Institute of Physiology, University of Oslo, for valuable discussions. We are also indebted to Jorunn Line Vaaland for sectioning, to Dr. F. Walvig, Biological Station, University of Oslo, Drobak, for supply of hagfishes, and to Dr. L. WaliSe, Institute of Physics, University of Oslo, for aid with the statistics. i Cell Tiss. Res. 157
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this d e p o l a r i z a t i o n causes release of Ca++-ions s t o r e d in t h e sarcoplasmic r e t i c u l u m during rest ( R i d g e w a y a n d Ashley, 1967), t h u s triggering contraction. The v e r y high m e m b r a n e c a p a c i t a n c e of muscle fibers (Katz, 1948) a p p e a r s a m p l y e x p l a i n e d b y t h e presence of the T - s y s t e m ( F a l k a n d F a t t , 1964). If t h e specific c a p a c i t a n c e of muscle fiber p l a s m a m e m b r a n e a n d T - t u b u l e m e m b r a n e is similar t o t h a t of u n m y e l i n a t e d axons, i.e. 1 ~zF/cm 2 (Hodgkin etal., 1952; Cole, 1968), t h e surface a r e a of t h e T - s y s t e m m u s t be several t i m e s t h e surface a r e a of t h e muscle fiber. E x a c t e v a l u a t i o n of t h e dimensions of t h e T - s y s t e m was first carried o u t in " w h i t e " fast t w i t c h fibers of frog's m. sartorius where t r i a d s occur r e g u l a r l y a t t h e Z-disks (Peachey, 1965). N a g (1972) has d e t e r m i n e d surface a n d volume of t h e T - s y s t e m t u b u l e s in b o t h " r e d " a n d " w h i t e " fibers of a teleost, with t h e t r i a d s still a t t h e Z-disks. The e s t i m a t e s of t h e T - s y s t e m area in these a n i m a l s fit quite well with r e p o r t s of m e m b r a n e c a p a c i t a n c e s ( F a l k a n d F a t t , 1964; H i d a k a a n d Toida, 1969; H o d g k i n a n d N a k a j i m a , 1972 a, b). I n a d d i t i o n , Luff a n d A t w o o d (1971, 1972) have m e a s u r e d t h e T - s y s t e m a r e a a n d c a p a c i t a n c e of b o t h slow t w i t c h a n d fast t w i t c h muscle fibers of t h e mouse, where t r i a d s are s i t u a t e d at A/I-junctions, a n d good a g r e e m e n t was f o u n d b e t w e e n m o r p h o l o g y a n d electrical properties. The p a r i e t a l muscle of hagfish consists of e n t i r e l y d i s t i n c t fast ( " w h i t e ") a n d slow ( " r e d " ) muscle fibers (Andersen et al., 1963; Alnms et al., 1964; Nicolaysen, 1964, 1966). I t was possible t o o b t a i n extensive d a t a on t h e complex i m p e d a n c e of b o t h these fiber t y p e s (Nicolaysen, 1974 a, b). I t s i n t e r p r e t a t i o n r e q u i r e d i n f o r m a t i o n on t h e m o r p h o l o g y of t h e T - s y s t e m in hagfish muscle fibers. This is p r e s e n t e d for four fiber t y p e s in this p a p e r , which also c o n t r i b u t e s to e x p l a i n differences in t h e e x c i t a t i o n - c o n t r a c t i o n coupling of slow a n d fast muscle fibers.
Material and Methods Atlantic hagfishes (Myxine glutinosa, L.) were used after storage for a few days in oxygenated seawater at 4~ Small samples of m. parietalis and m. craniovelaris were rapidly dissected out after decapitation of the animal, and generally fixed at room temperature by immersion overnight in 2% paraformaldehyde -k2.5 % glutaraldehyde in 0.6 M sodium cacodylate buffer ( ~ approximately isoosmotic), pH 7.2. During fixation the muscle samples were stretched to in vivo length, determined by measuring myotomal width. After dividing the samples into suitable smaller blocks and rinse in cacodylate buffer for 1 hr, the blocks were postfixed in 1% OsO4 for 1 hr, dehydrated in acetone and embedded with TAAB Embedding Kit (TK-3). Semithin sections were stained with p-phenylene-diamine (Korneliussen, 1972; Korneliussen and Nicolaysen, 1973). Ultrathin sections oriented transversely and longitudinally were stained with lead citrate. The material was examined with a Siemens Elmiskop I. Additional material from one specimen was fixed with 0.15, 0.30, 0.60 and 0.80 M sodium cacodylate as buffers. Two specimens were finally used for the complete quantitative studies of m. parietalis fibers, one small specimen 19 cm long (largest cross-sectional dimensions 0,7 • 0.9 cm, myotomal width 0.20 cm) and one large specimen 34 cm long (largest cross-sectional dimensions 1.6 • 1.8 cm, myotomal width 0.50 cm). A specimen with average size, 25 cm, was used for control estimation of density of triads as well as for quantitative studies of m. craniovelaris. In addition, native diameters and diameters in semithin sections were determined for different muscle fiber types in also a 26 cm long specimen (myotomal width 0.29 cm) and a 38 cm (myotomal width 0.43 cm) one. The methods used for evaluation of various quantitative parameters are referred in proper sections below.
Hagfish T-system
3
Results
Density o/Triads and T-tubules Fig. 1 displays the triad conformations relative to the sarcomeres 1 in a " white" muscle fiber of m. parietalis in Myxine. As reported earlier (Korneliussen and Nicolaysen, 1973) triads occur close to myofibrils in longitudinal sections. When muscles are stretched to in vivo length before fixation, the triads are located at A/I-junctions in all four muscle fiber types, i.e. " w h i t e " , "intermediate", and the " r e d " fibers of m. parietalis and " r e d " fibers of m. craniovelaris. Typical for all four fiber types is an irregular distribution of triads, since a number of A/Ijunctions lack triads. Furthermore, it is readily evident that the four muscle fiber types differ with regard to the density of triads (Korneliussen and Nicolaysen 1973). Any difference in the density of triads between deep and superficial portions of a particular fiber was never noted. The fact that some A/I-junctions lack triads indicates that the myofibrils are not completely surrounded by the T-tubules. When myofibrils occur regularly stacked with sarcomeres of neighbouring fibrils in phase, triads are apparently " s h a r e d " between myofibrils. Usually it is then not possible to assign a given triad to a particular myofibril. When myofibrils are stacked out of phase or when a large intermyofibrillar space intervenes between them, however, " e x t r a " triads may be found that apparently are not " s h a r e d " between adjacent myofibrils (Fig. 1 c, e, f). The first step in determining the dimensions of the T-system in Myxine muscle fibers was to obtain a measure for the density of triads. In order to cope with the irregular distribution of the triads and with the occurrence of the "extra" triads, triads were systematically counted relative to " s u b j a c e n t " sarcomeres in the electron micrographs unless they clearly could be assigned to an "overlying" sarcomere due to location ( " e x t r a " triads). Thus, each sarcomere possessed either none (alternative a), one (alternative b), or two {alternative d) triads, and in addition sometimes one (alternatives c and e) or two (alternative ]) " e x t r a " underlying ones. These alternatives a - / a r e illustrated in Figs. la-f, respectively, and in the left column of Table 1. Table 1 shows the occurrence (%) of the different alternatives of triads relative to sarcomeres in different muscle fiber types in three fishes. The number of sarcomeres (n) examined is indicated for each fiber type. There are consistent differences with regard to the various alternatives of triad/sarcomere arrangement in different fiber types. Most strikingly, sarcomeres without triads in longitudinal sections (alternative a) occur seldom in " w h i t e " fibers whereas they are numerous in the " r e d " ones. Furthermore, sarcomeres with two triads (alternative d) are more common in " w h i t e " fibers than in the " r e d " ones. Rather similar distributions are found in the three fishes in spite of the large difference in body size. From these counts the average number of triads and T-tubules per sectioned sarcomere (t) is calculated (Table 1). This value indicates to what extent the myofibrils are encircled by the tubules. If for example alternative / in Table 1 occurred consistently (i.e. t-~ 4) all myofibrils would be fully encircled twice per sarcomere by independent tubules. Correspondingly, alternative d at 100% would 1 The portion of a myofibril between two adjacent Z-disks. 1"
Fig. 1 a - - f . Longitudinal sections of sarcomeres from a " w h i t e " muscle fiber of m. parietalis. Black triangles indicate triads. (a-f) show triad conformations relative to sectioned sarcomeres t h a t correspond with alternatives a-/in Table 1. • 25000
Hagfish T-system
5
Table 1. Density of triads (%) relative to longitudinally sectioned sarcomeres in different muscle fiber types in three hagfishes. The mean number of triads per sectioned sarcomere (t) is given for all fiber types. A Fisher-Irwin test showed highly significant difference between t of "white" and "red" fibers for all specimens Location of triads relative to "subjacent" sareomeres
I
Small specimen (19 cm)
"Average specimen" (25 cm)
W %
W %
3.1
I %
R %
1 2 . 5 23.1
2.7
I %
R %
Large specimen (34 cm)
Cr. vel. W % %
I %
1~ %
1 3 . 2 23.6
25.2
4.9
1 4 . 1 19.6
[]
I []
I [] [] [] d
29.8
48.9
41.3
34.3
51.6
41.9
45.7
40.6
52.4
46.1
2.8
1.2
3.3
2.7
1.1
1.8
4.9
2..5
2.7
3.5
3 4 . 3 28.2
57.6
3 4 . 1 32..7 23.9
50.5
29.6
28..7
[] []
i
58.6
L
3.1
2.5
4.1
2.1
0.0
0.0
0.3
1.5
1.2
1.4
I
2.6
0.6
0.0
0.6
0.0
0.0
0.0
0.0
0.0
0.7
[] [] [] [] [] [] [] (353) (327) (269)
n Mean number of triads (T-tubules) per sectioned sarcomere (t)
1.72
1.30
1.17
( 3 3 0 ) (91) (110) ( 3 0 2 ) 1.64
1 . 2 2 1 . 1 1 1.04
( 2 0 2 ) (137) (143) 1.51
1 . 2 1 1.18
m e a n t h a t all myofibrils were also completely encircled twice per sarcomere, b u t the t u b u l e s would t h e n be shared b y a d j a c e n t myofibrils. T - t u b u l e s opening at the plasma m e m b r a n e were seen only occasionally, a n d accordingly the distance b e t w e e n the openings m u s t be r a t h e r great. I n m. craniovelaris single t u b u l e s r u n n i n g t r a n s v e r s e l y m a y belong to the T - s y s t e m (Korneliussen a n d Nicolaysen, 1973), b u t such t u b u l e s have n o t been included in the table. Occasional dyads are included.
Course and Possible Branching o/the T-tubules Possible b r a n c h i n g or i n t e r c o n n e c t i o n s between T - t u b u l e s m a y affect its efficiency in m e d i a t i n g electrical signals (Nicolaysen, 1974a). W e have looked s y s t e m a t i c a l l y for t u b u l a r b r a n c h i n g i n a large n u m b e r of both l o n g i t u d i n a l l y a n d t r a n s v e r s e l y oriented micrographs. B r a n c h i n g was n o t observed a n d we conclude t h a t the T - t u b u l e s in the hagfish r u n as separate a n d i n d e p e n d e n t units.
Size o/T-tubules Cross-sectioned T - t u b u l e s appear as elliptical profiles (Fig. 1; Figs. 6, 9, 11, 15 of K o r n e l i u s s e n a n d Nicolaysen, 1973). I n e s t i m a t i n g t h e i r cross-sectional area a n d
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H. Korneliussen and K. Nicolaysen
circumference (Table 2) the formulas applicable for ellipses were used. In the three muscle fiber types of m. parietalis the tubules appear similarly flattened and of approximately the same size whereas they are more circular in the m. craniovelaris fibers. The long and the short diameter of 10 T-tubule cross-sections from each fiber type were measured with a micrometer ocular on x 60000 prints. The measurements were taken from regions where the myofibrils appeared wide and thus probably were sectioned nearly axially, to ensure true cross-sections of the tubules and not oblique ones. The blocks of muscle tissue fixed in buffers of varying osmolarities (0.15-0.8 M) showed identical diameters of the T-tubules. The dimensions of the T-tubules therefore seem resistant to shrinkage or swelling during the fixation and embedding procedure. This stands in contrast to the dramatic effects on the T-tubules when they are exposed to varying osmolarities prior to fixation (Rapaport et al., 1969). This difference can probably be explained by time factors. During fixation the aldehydes probably diffuse rapidly and m a y stabilize the membranes before any osmotic influence takes place.
Sarcomere Length In the muscle tissue stretched to in vivo length before and during fixation the sarcomere length varied regionally between 2.1 and 2.6 ~zm without any significant difference between fiber types or between animals of different size. The mean was 2.4 ~zm and this value is used throughout the calculations. Size o/ the Myo/ibrils The circumference of the myofibrils was measured with a map measure and the cross-sectional area with a planimeter on transversely oriented x60000 prints (Table 2). The cross-sections vary from nearly circular to very elongated irregular profiles. However, most of the myofibrils possess circular to quadratic profiles. This is revealed in Table 2, as the circumference of the myofibrils is close to four times the square root of the cross-sectional area. The three fiber types of m. parietalis possess myofibrils of rather similar size (Figs. 2-3), whereas those of m. craniovelaris are much smaller (Fig. 4). Interestingly, the myofibrils of the small (Figs. 2a, 3a) and large (Figs. 2b, 3b) specimens possess nearly identical sizes. This suggests t h a t increase in muscle fiber diameter during growth involves an increase in the number of myofibrils rather than an increase in size of the individual myofibrils. The possibility that growth also involves increased intermyofibrillar space is apparently not supported by the electron micrographs (Figs. 2, 3). However, as will be shown below, the electron micrographs m a y be misleading in this respect. Muscle Fiber Diameter Muscle fiber diameters were calculated as d ~ 89189 where the cross-sectional areas of the fibers (A) were measured by weighing fiber projections from semithin sections (Table 2). The native diameters of a number of fibers stretched to resting length were also measured in vivo, when under the dissecting microscope (Table 2). These measurements were done on fibers in a myotome neighbouring the one used
Hagfish T-system
7
Fig. 2 a and b. Transverse sections of m. parietalis " w h i t e " fibers from a 19 cm long (a) and a 34 cm long (b) hagfish. Note the identical size of the myofibrils. No intermyofibrillar space. • 20 000
8
H. Korneliussen and K. Nicolaysen
Fig. 3 a and b. Transverse sections of " r e d " muscle fibers of m. parietalis of a 19 cm long (a) and a 34 cm long (b) hagfish. Note the identical size of the myofibrils, and the amount of intermyofibrillar space. )
Distribution and Dimension of the T-System in Different Muscle Fiber Types in the Atlantic Hagfish (Myxine glutinosa, L.) H e l g e K o r n e l i u s s e n a n d K ~ r e Nicolaysen* Anatomical Institute and Institute of Neurophysiology, University of 0slo, 0slo, Norway Received December 16, 1974
Summary. Triad density relative to sarcomeres, size of T-system tubules, sarcomere length, muscle fiber diameter in native and fixed states, and size of myofibrils were measured in four striated muscle fiber types in Atlantic hagfishes (Myxine glutinosa, L.) of different sizes. Triads occur at A/I-junctions in all fiber types. The density of triads relative to sarcomeres is higher in "white" than in " r e d " muscle fibers. The T-tubules show no sign of branching. The area of the T-system tubules is 3 4 times the surface area in 80 ~m "white" muscle fibers and 1-2 times that in 60 ~m " r e d " fibers. The size of myofibrils is similar in "white", "intermediate", and " r e d " fibers of m. parietalis, and constant through a large span of animal size. In "white" fibers, increase in diameter up to 90 ~zm is accompanied by an increase in the number of myofibrils, not by an increase in the individual size of the myofibrils. Above 90 ~m," white" fibers grow by increasing the amount of intermyofibrillar space. This is reflected by an extensive shrinkage of the thicker "white" fibers during the preparative procedure for electron microscopy, a shrinkage that is limited only by complete packing of the myofibrils. " R e d " fibers shrink much less. Key words: Muscle fiber types (Myxine glutinosa, L.) - - T - s y s t e m - G r o w t h - Shrinkage - - Electron microscopy. Introduction I n a previous p u b l i c a t i o n we defined four u l t r a s t r u c t u r a l l y different muscle fiber t y p e s in t h e A t l a n t i c hagfish (Myxine glutinosa, L.) (Korneliussen a n d Nicolaysen, 1973). R e c e n t i n v e s t i g a t i o n s b y one of us (Nicolaysen, 1974 a, b) p r o m p t e d a closer analysis of t h e T - s y s t e m in this animal. As r e v i e w e d b y S m i t h (1966) a n d F r a n c i n i - A r m s t r o n g (1973) t h e t r i a d s m e d i a t e t h e e x c i t a t i o n - c o n t r a c t i o n coupling in skeletal muscle fibers. T h e Ts y s t e m t u b u l e s (Andersson-Cedergren, 1959) form t h e central e l e m e n t s of t h e t r i a d s . T h e y are open a t t h e surface, a t least t o some e x t e n t , a n d t h u s t h e s y s t e m r e p r e s e n t s a g e o m e t r i c a l l y c o m p l i c a t e d e x t e n s i o n of t h e e x t r a c e l l u l a r space. T h e c o n t i n u i t y w i t h t h e e x t r a c e l l u l a r space has been d e m o n s t r a t e d b y t h e e n t r a n c e of e x t r a c e l l u l a r t r a c e r s into t h e tubules. T h e surface d e p o l a r i z a t i o n resulting from a c t i v i t y in t h e m o t o r axons is c o n d u c t e d i n w a r d s along t h e T - t u b u l e s . S o m e h o w
Send o]/print requests to: Dr. H. Korneliussen, Anatomical Institute, University of Oslo, Karl Johansgt. 47, Oslo 1, Norway. * We thank Dr. J. K. S. Jansen, Institute of Physiology, University of Oslo, for valuable discussions. We are also indebted to Jorunn Line Vaaland for sectioning, to Dr. F. Walvig, Biological Station, University of Oslo, Drobak, for supply of hagfishes, and to Dr. L. WaliSe, Institute of Physics, University of Oslo, for aid with the statistics. i Cell Tiss. Res. 157
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H. Korneliussen and K. Nicolaysen
this d e p o l a r i z a t i o n causes release of Ca++-ions s t o r e d in t h e sarcoplasmic r e t i c u l u m during rest ( R i d g e w a y a n d Ashley, 1967), t h u s triggering contraction. The v e r y high m e m b r a n e c a p a c i t a n c e of muscle fibers (Katz, 1948) a p p e a r s a m p l y e x p l a i n e d b y t h e presence of the T - s y s t e m ( F a l k a n d F a t t , 1964). If t h e specific c a p a c i t a n c e of muscle fiber p l a s m a m e m b r a n e a n d T - t u b u l e m e m b r a n e is similar t o t h a t of u n m y e l i n a t e d axons, i.e. 1 ~zF/cm 2 (Hodgkin etal., 1952; Cole, 1968), t h e surface a r e a of t h e T - s y s t e m m u s t be several t i m e s t h e surface a r e a of t h e muscle fiber. E x a c t e v a l u a t i o n of t h e dimensions of t h e T - s y s t e m was first carried o u t in " w h i t e " fast t w i t c h fibers of frog's m. sartorius where t r i a d s occur r e g u l a r l y a t t h e Z-disks (Peachey, 1965). N a g (1972) has d e t e r m i n e d surface a n d volume of t h e T - s y s t e m t u b u l e s in b o t h " r e d " a n d " w h i t e " fibers of a teleost, with t h e t r i a d s still a t t h e Z-disks. The e s t i m a t e s of t h e T - s y s t e m area in these a n i m a l s fit quite well with r e p o r t s of m e m b r a n e c a p a c i t a n c e s ( F a l k a n d F a t t , 1964; H i d a k a a n d Toida, 1969; H o d g k i n a n d N a k a j i m a , 1972 a, b). I n a d d i t i o n , Luff a n d A t w o o d (1971, 1972) have m e a s u r e d t h e T - s y s t e m a r e a a n d c a p a c i t a n c e of b o t h slow t w i t c h a n d fast t w i t c h muscle fibers of t h e mouse, where t r i a d s are s i t u a t e d at A/I-junctions, a n d good a g r e e m e n t was f o u n d b e t w e e n m o r p h o l o g y a n d electrical properties. The p a r i e t a l muscle of hagfish consists of e n t i r e l y d i s t i n c t fast ( " w h i t e ") a n d slow ( " r e d " ) muscle fibers (Andersen et al., 1963; Alnms et al., 1964; Nicolaysen, 1964, 1966). I t was possible t o o b t a i n extensive d a t a on t h e complex i m p e d a n c e of b o t h these fiber t y p e s (Nicolaysen, 1974 a, b). I t s i n t e r p r e t a t i o n r e q u i r e d i n f o r m a t i o n on t h e m o r p h o l o g y of t h e T - s y s t e m in hagfish muscle fibers. This is p r e s e n t e d for four fiber t y p e s in this p a p e r , which also c o n t r i b u t e s to e x p l a i n differences in t h e e x c i t a t i o n - c o n t r a c t i o n coupling of slow a n d fast muscle fibers.
Material and Methods Atlantic hagfishes (Myxine glutinosa, L.) were used after storage for a few days in oxygenated seawater at 4~ Small samples of m. parietalis and m. craniovelaris were rapidly dissected out after decapitation of the animal, and generally fixed at room temperature by immersion overnight in 2% paraformaldehyde -k2.5 % glutaraldehyde in 0.6 M sodium cacodylate buffer ( ~ approximately isoosmotic), pH 7.2. During fixation the muscle samples were stretched to in vivo length, determined by measuring myotomal width. After dividing the samples into suitable smaller blocks and rinse in cacodylate buffer for 1 hr, the blocks were postfixed in 1% OsO4 for 1 hr, dehydrated in acetone and embedded with TAAB Embedding Kit (TK-3). Semithin sections were stained with p-phenylene-diamine (Korneliussen, 1972; Korneliussen and Nicolaysen, 1973). Ultrathin sections oriented transversely and longitudinally were stained with lead citrate. The material was examined with a Siemens Elmiskop I. Additional material from one specimen was fixed with 0.15, 0.30, 0.60 and 0.80 M sodium cacodylate as buffers. Two specimens were finally used for the complete quantitative studies of m. parietalis fibers, one small specimen 19 cm long (largest cross-sectional dimensions 0,7 • 0.9 cm, myotomal width 0.20 cm) and one large specimen 34 cm long (largest cross-sectional dimensions 1.6 • 1.8 cm, myotomal width 0.50 cm). A specimen with average size, 25 cm, was used for control estimation of density of triads as well as for quantitative studies of m. craniovelaris. In addition, native diameters and diameters in semithin sections were determined for different muscle fiber types in also a 26 cm long specimen (myotomal width 0.29 cm) and a 38 cm (myotomal width 0.43 cm) one. The methods used for evaluation of various quantitative parameters are referred in proper sections below.
Hagfish T-system
3
Results
Density o/Triads and T-tubules Fig. 1 displays the triad conformations relative to the sarcomeres 1 in a " white" muscle fiber of m. parietalis in Myxine. As reported earlier (Korneliussen and Nicolaysen, 1973) triads occur close to myofibrils in longitudinal sections. When muscles are stretched to in vivo length before fixation, the triads are located at A/I-junctions in all four muscle fiber types, i.e. " w h i t e " , "intermediate", and the " r e d " fibers of m. parietalis and " r e d " fibers of m. craniovelaris. Typical for all four fiber types is an irregular distribution of triads, since a number of A/Ijunctions lack triads. Furthermore, it is readily evident that the four muscle fiber types differ with regard to the density of triads (Korneliussen and Nicolaysen 1973). Any difference in the density of triads between deep and superficial portions of a particular fiber was never noted. The fact that some A/I-junctions lack triads indicates that the myofibrils are not completely surrounded by the T-tubules. When myofibrils occur regularly stacked with sarcomeres of neighbouring fibrils in phase, triads are apparently " s h a r e d " between myofibrils. Usually it is then not possible to assign a given triad to a particular myofibril. When myofibrils are stacked out of phase or when a large intermyofibrillar space intervenes between them, however, " e x t r a " triads may be found that apparently are not " s h a r e d " between adjacent myofibrils (Fig. 1 c, e, f). The first step in determining the dimensions of the T-system in Myxine muscle fibers was to obtain a measure for the density of triads. In order to cope with the irregular distribution of the triads and with the occurrence of the "extra" triads, triads were systematically counted relative to " s u b j a c e n t " sarcomeres in the electron micrographs unless they clearly could be assigned to an "overlying" sarcomere due to location ( " e x t r a " triads). Thus, each sarcomere possessed either none (alternative a), one (alternative b), or two {alternative d) triads, and in addition sometimes one (alternatives c and e) or two (alternative ]) " e x t r a " underlying ones. These alternatives a - / a r e illustrated in Figs. la-f, respectively, and in the left column of Table 1. Table 1 shows the occurrence (%) of the different alternatives of triads relative to sarcomeres in different muscle fiber types in three fishes. The number of sarcomeres (n) examined is indicated for each fiber type. There are consistent differences with regard to the various alternatives of triad/sarcomere arrangement in different fiber types. Most strikingly, sarcomeres without triads in longitudinal sections (alternative a) occur seldom in " w h i t e " fibers whereas they are numerous in the " r e d " ones. Furthermore, sarcomeres with two triads (alternative d) are more common in " w h i t e " fibers than in the " r e d " ones. Rather similar distributions are found in the three fishes in spite of the large difference in body size. From these counts the average number of triads and T-tubules per sectioned sarcomere (t) is calculated (Table 1). This value indicates to what extent the myofibrils are encircled by the tubules. If for example alternative / in Table 1 occurred consistently (i.e. t-~ 4) all myofibrils would be fully encircled twice per sarcomere by independent tubules. Correspondingly, alternative d at 100% would 1 The portion of a myofibril between two adjacent Z-disks. 1"
Fig. 1 a - - f . Longitudinal sections of sarcomeres from a " w h i t e " muscle fiber of m. parietalis. Black triangles indicate triads. (a-f) show triad conformations relative to sectioned sarcomeres t h a t correspond with alternatives a-/in Table 1. • 25000
Hagfish T-system
5
Table 1. Density of triads (%) relative to longitudinally sectioned sarcomeres in different muscle fiber types in three hagfishes. The mean number of triads per sectioned sarcomere (t) is given for all fiber types. A Fisher-Irwin test showed highly significant difference between t of "white" and "red" fibers for all specimens Location of triads relative to "subjacent" sareomeres
I
Small specimen (19 cm)
"Average specimen" (25 cm)
W %
W %
3.1
I %
R %
1 2 . 5 23.1
2.7
I %
R %
Large specimen (34 cm)
Cr. vel. W % %
I %
1~ %
1 3 . 2 23.6
25.2
4.9
1 4 . 1 19.6
[]
I []
I [] [] [] d
29.8
48.9
41.3
34.3
51.6
41.9
45.7
40.6
52.4
46.1
2.8
1.2
3.3
2.7
1.1
1.8
4.9
2..5
2.7
3.5
3 4 . 3 28.2
57.6
3 4 . 1 32..7 23.9
50.5
29.6
28..7
[] []
i
58.6
L
3.1
2.5
4.1
2.1
0.0
0.0
0.3
1.5
1.2
1.4
I
2.6
0.6
0.0
0.6
0.0
0.0
0.0
0.0
0.0
0.7
[] [] [] [] [] [] [] (353) (327) (269)
n Mean number of triads (T-tubules) per sectioned sarcomere (t)
1.72
1.30
1.17
( 3 3 0 ) (91) (110) ( 3 0 2 ) 1.64
1 . 2 2 1 . 1 1 1.04
( 2 0 2 ) (137) (143) 1.51
1 . 2 1 1.18
m e a n t h a t all myofibrils were also completely encircled twice per sarcomere, b u t the t u b u l e s would t h e n be shared b y a d j a c e n t myofibrils. T - t u b u l e s opening at the plasma m e m b r a n e were seen only occasionally, a n d accordingly the distance b e t w e e n the openings m u s t be r a t h e r great. I n m. craniovelaris single t u b u l e s r u n n i n g t r a n s v e r s e l y m a y belong to the T - s y s t e m (Korneliussen a n d Nicolaysen, 1973), b u t such t u b u l e s have n o t been included in the table. Occasional dyads are included.
Course and Possible Branching o/the T-tubules Possible b r a n c h i n g or i n t e r c o n n e c t i o n s between T - t u b u l e s m a y affect its efficiency in m e d i a t i n g electrical signals (Nicolaysen, 1974a). W e have looked s y s t e m a t i c a l l y for t u b u l a r b r a n c h i n g i n a large n u m b e r of both l o n g i t u d i n a l l y a n d t r a n s v e r s e l y oriented micrographs. B r a n c h i n g was n o t observed a n d we conclude t h a t the T - t u b u l e s in the hagfish r u n as separate a n d i n d e p e n d e n t units.
Size o/T-tubules Cross-sectioned T - t u b u l e s appear as elliptical profiles (Fig. 1; Figs. 6, 9, 11, 15 of K o r n e l i u s s e n a n d Nicolaysen, 1973). I n e s t i m a t i n g t h e i r cross-sectional area a n d
6
H. Korneliussen and K. Nicolaysen
circumference (Table 2) the formulas applicable for ellipses were used. In the three muscle fiber types of m. parietalis the tubules appear similarly flattened and of approximately the same size whereas they are more circular in the m. craniovelaris fibers. The long and the short diameter of 10 T-tubule cross-sections from each fiber type were measured with a micrometer ocular on x 60000 prints. The measurements were taken from regions where the myofibrils appeared wide and thus probably were sectioned nearly axially, to ensure true cross-sections of the tubules and not oblique ones. The blocks of muscle tissue fixed in buffers of varying osmolarities (0.15-0.8 M) showed identical diameters of the T-tubules. The dimensions of the T-tubules therefore seem resistant to shrinkage or swelling during the fixation and embedding procedure. This stands in contrast to the dramatic effects on the T-tubules when they are exposed to varying osmolarities prior to fixation (Rapaport et al., 1969). This difference can probably be explained by time factors. During fixation the aldehydes probably diffuse rapidly and m a y stabilize the membranes before any osmotic influence takes place.
Sarcomere Length In the muscle tissue stretched to in vivo length before and during fixation the sarcomere length varied regionally between 2.1 and 2.6 ~zm without any significant difference between fiber types or between animals of different size. The mean was 2.4 ~zm and this value is used throughout the calculations. Size o/ the Myo/ibrils The circumference of the myofibrils was measured with a map measure and the cross-sectional area with a planimeter on transversely oriented x60000 prints (Table 2). The cross-sections vary from nearly circular to very elongated irregular profiles. However, most of the myofibrils possess circular to quadratic profiles. This is revealed in Table 2, as the circumference of the myofibrils is close to four times the square root of the cross-sectional area. The three fiber types of m. parietalis possess myofibrils of rather similar size (Figs. 2-3), whereas those of m. craniovelaris are much smaller (Fig. 4). Interestingly, the myofibrils of the small (Figs. 2a, 3a) and large (Figs. 2b, 3b) specimens possess nearly identical sizes. This suggests t h a t increase in muscle fiber diameter during growth involves an increase in the number of myofibrils rather than an increase in size of the individual myofibrils. The possibility that growth also involves increased intermyofibrillar space is apparently not supported by the electron micrographs (Figs. 2, 3). However, as will be shown below, the electron micrographs m a y be misleading in this respect. Muscle Fiber Diameter Muscle fiber diameters were calculated as d ~ 89189 where the cross-sectional areas of the fibers (A) were measured by weighing fiber projections from semithin sections (Table 2). The native diameters of a number of fibers stretched to resting length were also measured in vivo, when under the dissecting microscope (Table 2). These measurements were done on fibers in a myotome neighbouring the one used
Hagfish T-system
7
Fig. 2 a and b. Transverse sections of m. parietalis " w h i t e " fibers from a 19 cm long (a) and a 34 cm long (b) hagfish. Note the identical size of the myofibrils. No intermyofibrillar space. • 20 000
8
H. Korneliussen and K. Nicolaysen
Fig. 3 a and b. Transverse sections of " r e d " muscle fibers of m. parietalis of a 19 cm long (a) and a 34 cm long (b) hagfish. Note the identical size of the myofibrils, and the amount of intermyofibrillar space. )
