JOURNAL

OF U L T R A S T R U C T U R E

RESEARCH

55, 212-227

(1976)

Ultrastructural Differentiation of Skeletal Muscle Fibers and Thei~ Diversity ROBERT J . TOMANEK

Department of Anatomy, College of Medicine, University of Iowa, Iowa City, Iowa 52242 Received September 9, 1975, and in revised form, January 14, 1976 The u l t r a s t r u c t u r a l changes occurring during postnatal differentiation of the plantaris and soleus muscles were investigated in kittens. Neonatal muscle fibers vary somewhat according to m a t u r i t y as well as Z-line width and relative volume of sareoplasmic reticulum (variables most characteristic of a given fiber type). A clear-cut classification of fiber type, however, evolves during the postnatal p e r i o d - i n t e r m e d i a t e fibers differentiate first while white fibers differentiate last. Functional overload, induced by ablation of the synergistic gastrocnemius, does not significantly alter the prime characteristics of fiber type (e.g., Z-line width) but r a t h e r exerts a n influence on accelerating myofibril proliferation, which occurs both by splitting and by de novo formation. White fibers are modified by enhancing t h e i r mitochondrial population. These results support previous histochemical studies and affirm the contention t h a t certain fiber characteristics (structural and enzymatic) are determined genetically and expressed through neural factors, while other characteristics are subject to modification by alterations in function.

Skeletal muscle fibers of most mammalian species undergo differentiation during postnatal growth. This process is exemplified by changes in the contractile properties of various limb muscles (8, 11, 29), changes in myosin ATPase, and the diversification of fibers according to histochemical characteristics (1, 12, 13, 34, 38, 52, 60). Although ultrastructural changes have been described with reference to selected muscles (22, 25, 48, 59) and the tubular system of fibers from fast and slow muscles compared (36), the ultrastructural differentiation of specific fiber types has not been defined. It is well known that muscle fibers in adult mammals are diversifted not only according to histochemical criteria, but ultrastructural characteristics as well (23, 40, 42, 53). The present study was initiated as a corollary to recent work in this laboratory which defined the time course and specific histochemical characteristics of the fibers comprising the fast-twitch plantaris and slow-twitch soleus muscles of the kitten during postnatal differentiation (52). Accordingly, this work was concerned with

determining: (1) whether certain phenotypic ultrastructural features are evident in histochemically undifferentiated fibers, (2) whether specific modes of differentiation may typify different types of fibers, and (3) whether ultrastructural differentiation corresponds to the histochemical characteristics of a given fiber. In order to define the time course and nature of differentiation more clearly quantitative data concerning the sarcoplasmic reticulum and Z-lines are presented. Since some factor(s) associated with compensatory hypertrophy accelerates the maturation process in these muscles (52), the role of functional overload was again investigated. Because of the diverse nomenclature used to categorize three basic types of mammalian twitch fibers, considerable confusion still exists regarding the specific characteristics of a given population of fibers. This paper utilizes a common, albeit nondescriptive nomenclature, which is well established in the literature (16, 39, 41,53). "Red" fibers are rich in large mitochondria and have ample sarcoplasmic reticulum and moderately wide Z-lines, 212

Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

213

SKELETAL MUSCLE FIBER DIVERSITY

while ¢~white" fibers contain a sparse mitochondrial population, an extensive sarcoplasmic reticulum, and thin Z-lines. "Intermediate" fibers, typical of the mammalian soleus, h a v e fairly numerous mitochondria (but smaller than those found in red fibers), scanty sarcoplasmic reticulum, numerous lipid inclusions, and wide Zlines. Other examples of nomenclature for red, white, and intermdiate, respectively, include: (1) fast-twitch-oxydative-glycolytic, fast-twitch-glycolytic, and slow-twitch oxidative (43); (2), C, A, and B (51); and (3) IIA, IIB, and I (6). MATERIALS AND METHODS Specimens, excised from the midbelly of the plantaris and soleus muscles of 20 kittens of various ages and two adult cats were tied to wooden splints and fixed in a solution consisting of 3% glutaraldehyde, 0.8% paraformaldehyde, 0.02 M CaC12, and 0.1 M sodium cacodylate. They were postfixed in 1% osmium tetroxide, dehydrated in alcohol, and embedded in Epon. Thin sections were cut with a diamond knife on a P o r t e r - B l u m MT-2 microtome, stained with uranyl acetate and lead citrate, and examined with a Siemens Elmiscope 101. Compensatory hypertrophy of the plantaris and soleus muscles of the r i g h t hind-limb in six neonatal kittens was induced (under ether anesthesia) by extirpation of the synergistic gastrocnemius. These animals were sacrificed at 3 or 6 weeks of age. Such functional overload is associated with significant weight increases of both muscles due to hypertrophy of their various fiber type populations (52). A measuring magnifier (micrometer scale in 0. lm m divisions; magnification, x8) was employed to measure Z-line width directly on micrographs (x30 000) of longitudinally sectioned fibers. Approximately nine Z-lines per cell were measured and the mean was recorded. Due to irregularities within any Z-line it was essential to establish criteria for estimating this parameter. Because the dense fuzzy matrix at the borders of the Z-line presents some problems (18), we measured Z-line regions which were consistent in width, and excluded the outer, irregular (serrated) regions of the Z-line which consist of thickened portions of t h i n filaments. By having two investigators measure the same Z-lines (N = 25) we found the m e a n of the differences to be 9%. The ratio of sarcoplasmic reticulum/myofibril was determined from micrographs (x 30 000) of cross-sectional fields. A double test grid with intercepts every 2.5 m m was randomly superimposed over the micrograph and the n u m b e r of intercepts

falling on a prescribed region of contractile material sectioned through either the A- or I-band, were counted separately. The n u m b e r of points falling on the sarcoplasmic reticulum delineating this region were similarly counted and the volume ratio (%) of sarcoplasmic reticulum (Vsr) at the I-band (I) or Aband (A) was estimated by the relation:

v,~/v~ = p~/p, x lOO,

or V s r / V A = Psr/PA × 100

where P is the n u m b e r of points counted. Owing to the extensiveness and dilatations of the tubules at the Z-band, data from this sarcomere region were not included. We also excluded t e r m i n a l cisternae which were readily identified by t h e i r opaqueness. The point-counting technique was also used to estimate the relative mitochondrial volume in micrographs of longitudinally sectioned specimens. For the purpose of consistency subsarcolemmal regions were excluded. RESULTS

Muscle Fibers of the Neonate

Although the fibers comprising the soleus and plantaris muscles vary considerably with respect to the organization of subcellular components, they generally lack sufficiently distinctive features permitting classification of "fiber type." Where exceptions to this rule exist, the fiber resembles the intermediate type which constitutes virtually the total fiber population in the adult soleus. In both muscles various stages of development appear to be represented at birth (Figs. 3-6). Contractile filaments may be disoriented, the formation of sarcomeres incomplete, or where filaments are oriented, the peripheral regions of myofibrils may have scattered filaments. The presence of tubular elements and mitochondria appears to be related to the development of the contractile system, i.e., fibers with well-aligned contractile components have relatively well-defined organelles. The sarcoplasmic reticulum, even when present in large volume, lacks the orientation and distribution which typifies later stages. Distinct myotubes were occasionally observed, but

214

R O B E R T J. T O M A N E K

[ ] WHITE [ ] [NTERMED[ATE [ ] PLANTARIS [ ] RED [ ] UNDIFFERENTIATED [ ] SOLEUS (5) 140 :E _] :7 ]~(51

~" 120 -

." •

"

100 - • ": .:'.

""

• _~

'

6)

~

~. /,

(3: /

,..

u NEO-

NATAL

P / /

/

8o - : "

~:

/

(7)¢

5

' 6

10-12

ADULT

WEEKS

FIG. 1. Z-line w i d t h s . B a r s indicate g r o u p m e a n s ; t h e n u m b e r of fibers i n c l u d e d is g i v e n in p a r e n t h e ses; SE is indicated. V a l u e s for u n d i f f e r e n t i a t e d fibers (dots) are for s i n g l e fibers.

more commonly fibers with peripheral foci of sarcoplasm with scattered mitochondria, and glycogen were seen. In contrast to such relatively immature fibers, some cells have densely packed myofibrils with Z-lines in registry with those of adjacent myofibrils. Such fibers may contain a few triads which are not yet oriented; triad development appears to be independent of the remainder of the sarcoplasmic reticulure since the former lies isolated during this formative period. Most cells are uniformly small; mitochondria are narrow and lack orientation. Relative mitochondrial volume ranges between 3 and 9% (X +_ SE = 5.1 _+ 0.5%). Most nuclei lie in the cell periphery and many of these are components of satellite cells. Neonatal muscle cells are typified by lipid inclusions which persist in most fibers for some time following birth. Roughsurfaced membranes as well as free ribosomes and polyribosomes are abundant particularly in (1) peripheral cell regions, (2) the ends of the fiber, and (3) foci where sarcomere formation is evident. As these organelles relate to myofibril synthesis

during growth, they are described in the subsequent section. Quantitative data regarding Z-line width and the relative percentage of sarcoplasmic reticulum/myofibril volume are provided in Figs. ] and 2, respectively. It is evident that a wide range of values exist at birth for these parameters. However, with the exception of occasional fibers similar to the intermediate type of the adult, Z-line width and the extensiveness of the sarcoplasmic reticulum show no distinct relationship. This factor together with the apparent random distribution of mitochondria preclude the classification of fibers in the neonatal cat.

Growth and Differentiation While ultrastructural characteristics vary from fiber to fiber during the first 3

5

"-

F I

:

_:1.,

"'"

i o °



S

0 7

L~

:

A- BANO -

-

E~ WHITE []RED [ ] INTERMEDIATE

S '

I- BAND {lO)

T

4 ~

[4) ,

\

5

~d

2

o

"I

NEONATAL

(7)

P

6 WEEKS

P 10-12

)

[ p ~ ) ADULT

Fia. 2. R e l a t i v e v o l u m e of s a r c o p l a s m i c reticul u m (expressed as a ratio of S.R./myofibril at t h e A or I bands). Dots r e p r e s e n t i n d i v i d u a l v a l u e s for u n d i f f e r e n t i a t e d fibers w h i l e b a r s a r e g r o u p m e a n s ; t h e n u m b e r of fibers included is g i v e n in p a r e n t h e ses.

SKELETAL MUSCLE FIBER DIVERSITY

weeks of postnatal life, the fibers are still insufficiently differentiated according to the criteria previously described and therefore cannot be conveniently classified relative to fiber type (Figs. 10-15). It is, however, apparent that individual fibers in the plantaris vary considerably with regard to Z-line width, while the fibers of the soleus now contain a more homogeneous fiber population of Z-line widths (Fig. 1). Thus, the fibers of the soleus have Zline widths approaching those of older animals, and other ultrastructural characteristics begin to resemble those seen in the adult but are less clear cut (e.g., mitochondria frequently lack orientation at the Iband). The volume of the sarcoplasmic reticulum in both muscles is greater in the 3week-old kitten than in the neonatal specimens but is highly variable from fiber to fiber (Fig. 2). The increase in these values appears to be due to dilation rather than proliferation of the reticulum, which for the most part is loosely organized with the specializations typical of the A-band, Iband, and Z-line poorly defined. In 6-week-old kittens intermediate fibers appear to be well differentiated. A second type of fiber (plantaris) meets most of the criteria of the adult red type and contains Z-lines approximately 110 nm wide (Fig. 1). Fibers with more narrow Zlines (65-90 nm) are also evident but often contain fairly numerous mitochondria and therefore do not meet all of the criteria indicated for white fibers. In 10- to 12week-old kittens three populations of fibers can be identified qualitatively; moreover, their Z-line widths and volume of sarcoplasmic reticulum (quantitative

215

data) approach that of the adult. These data and observations suggest t h a t ultrastructural differentiation is completed first in intermediate, then in red, and finally in white fibers. The red and white fibers appear to have similar characteristics during the first 6 weeks of differentiation. White fibers become distinguishable as their mitochondrial content decreases substantially during the second month of life and the sarcoplasmic reticulum becomes organized and extensive. Figures 79 illustrate some typical changes in the reticulum between 3 and 10 weeks in fibers of the plantaris. The dilatated reticulum, characteristic of fibers from 3-week-old animals, is reduced in volume by 6 weeks, although it becomes more specialized with distinct connections with triads (Fig. 20). By 10 weeks of age the reticulum is extensive, highly organized, and typical of the adult. These observations are supported by the quantitative data (Fig. 2). The various types of fibers characteristic of the adult are illustrated in Figs. 24-27. Differences in the relative mitochondrial volume for the three fiber types are most evident in the adult; mean values _+ SE are: 6.7 _+ 1.5, 3.7 _+ 0.4, and 0.8 _+ 0.2% for red, intermediate, and white fibers, respectively. Evidence of filament synthesis, the formation of sarcomeres, and the enlargement of myofibrils is evident in all postnatal stages of development. A common sequence of sarcomere formation, however, is not apparent. New sarcomeres are commonly added to the ends of the myofibril providing growth in length, but during the rapid growth period (1-6 weeks) sarcomereogenesis may occur between adjacent

FIGs. 3-6. Neonatal muscle fibers at various stages of development. All figures × 20 00O. FIG. 3. Soleus. Disoriented filaments grouped in loci of myofibrils with small dense particles resembling ribosomes at peripheral.sites (arrowheads). mi, mitochondria. Fin. 4. Soleus. Well-oriented filaments assembled in myofibrils surrounded by mitochondria and sarcoplasmic reticulum (arrowheads). Lipid droplets (li) are evident in virtually all neonatal fibers. A, A-band; I, I-band; Z, Z-line. FIG. 5. Plantaris. Myofibril formation illustrating groups of filaments many of which are loosely ar: ranged. Fxo. 6. Plantaris. Well defined myofibrils, With sareoplasmic reticulum (arrowheads), and lipid inclusions. A portion of a satellite cell (SC) is included.

216

217

218

ROBERT J. TOMANEK

preexisting sarcomeres or between terminal points of two adjacent (end-to-end) myofibrils (Fig. 16). The assembly of thick and thin filaments in the p l a n t a r i s m a y precede the e s t a b l i s h m e n t of the Z-line (Fig. 17), but more commonly the Z-line is present while I-band development lags behind t h a t of the A-band (Fig. 18). The patt e r n of sarcomere formation in the soleus appears to show relative consistency. Thick and thin filaments lie disoriented a m o n g dense bodies of p r e s u m e d Z-line substance (Fig. 19). Such foci are n e a r l y always peripheral within the fiber and often perinuclear. Subsequently, the filaments and Z-lines m u s t align themselves to form a sarcomere. One often encounters myofibrils with s t r e a m i n g Z-line substance and s o m e w h a t well-aligned filam e n t s which a p p e a r to be more advanced stages of this process. The proliferation of myofibrils is m a r k e d d u r i n g early postnatal growth and appears to occur by (1) splitting of preexisting fibrils (Fig. 11) and (2) synthesis of filaments p r i m a r i l y in peripheral foci, but also between existing fibrils (Fig. 14). Myofibrils are not always clearly d e m a r c a t e d and m a y form a syncytium; this is the rule in i n t e r m e d i a t e type fibers as supported by observations on the soleus. Myofibrils increase t h e i r d i a m e t e r s by addition of newly synthesized filaments on t h e i r periphery where the filaments m a y lack the n o r m a l hexagonal spatial a r r a n g e m e n t and often lack orientation. Such sites, as well as the ends of myofibrils, contain small dense particles of similar size and density (often in clusters but also commonly associated with the r e t i c u l u m in

peripheral foci) along with disoriented contractile filaments.

Compensatory Hypertrophy Compensatory h y p e r t r o p h y d u r i n g postnatal growth is m a r k e d , and as previously noted, affects various fiber types in both the plantaris and soleus muscles (52). The u l t r a s t r u c t u r a l characteristics of some fibers from hypertrophic muscles (at 3 and 6 weeks) do not a p p e a r to differ q u a l i t a t i v e l y from those of control specimens with the exception t h a t the former contain a g r e a t e r n u m b e r of myofibrils. Fiber diversification, evident in control plantaris muscles at 6 weeks, is also evident in hypertrophic specimens with the exception t h a t the n u m b e r of mitochondria is relatively high in all fibers. However, the enhanced growth d u r i n g functional overload is manifest in a n u m b e r of u l t r a s t r u c t u r a l characteristics. First, in m a n y fibers t h e r e appear n u m e r o u s sites suggesting formation of new myofibrils-foci of scattered filaments with associated particles (ribosomes) and f r e q u e n t splitting of myofibrils (Fig. 21). Second, t h e r e occur fibers with disarranged sarcomeres, p a r t i c u l a r l y disoriented filaments in the I-band and s t r e a m i n g of Z-lines (Fig. 21). Such regions usually have ribosomes in close proximity and are suggestive of sarcomereogenesis. Third, fibers with myofibrils a r r a n g e d p e r p e n d i c u l a r to each other were also seen on occasion and a p p e a r to r e p r e s e n t highly accelerated growth (Fig. 22). The presence of myotubes, while uncommon, was noted in the hypertrophic plantaris (Fig. 23). While the widths of Z-lines in fibers

7-9. Development of the sarcoplasmic reticulum in plantaris fibers. All figures × 20 000. FIG. 7. An undifferentiated fiber (3 weeks postpartum). Reticulum is dilatated, but collars at Z-line (Z) are not yet well developed. All fibers contain numerous mitochondria at this age. I, I-band; A, A-band. Fro. 8. A fiber from a 6-week-oldanimal. The volume of the reticulum is reduced compared to the 3-week age group, but well-established collars of sarcoplasmic reticulum (arrows) are evident at the Z-line. This fiber appears to be differentiating into the white fiber type, as mitochondrial density is somewhat lower than that common to other fiber types. Transverse and oblique components of the sarcoplasmic reticulum (arrowheads) are commonly first seen at this stage. FIG. 9. A white fiber from a 10-week-old animal. Note the extensive development of the sarcoplasmic reticulum, well-defined myofibrils, and sparsity of mitochondria. FIGs.

SKELETAL MUSCLE FIBER DIVERSITY

from hypertrophic muscles were as diversifted as those from controls, the relative volume of the sarcoplasmic reticulum was increased in the plantaris red fibers following 6 weeks of functional overload. The values for this par a m et er at the A- and Ibands, respectively, are: 1.76 _+ 0.30 and 3.48 _+ 0.42% for hypertrophic red fibers compared to 0.90 ___0.13 and 1.66 +_ 0.23% for the controls. These differences are statistically significant (P < 0.05), but appear to be due, at least in part, to dilatation of the reticulum. Hypertrophic intermediate fibers in the soleus do not differ significantly from those of the controls (A- and Ibands, respectively): hypertrophic = 0.54 _+ 0.06 and 1.27 +__ 0.11; control = 0.48 +__ 0.08 and 1.01 _+ 0.15. DISCUSSION

Neonatal Fibers The results of the present study indicate that neonatal skeletal muscle fibers show considerable ultrastructural variation.

219

Some of the differences may be due to the state of maturation, e.g., alignment of illaments, sarcoplasmic volume, and mitochondrial density. While there is a lack of evidence demonstrating any consistent set of ultrastructural characteristics indicative of specific fiber types, the wide range of Z-line widths in different fibers is not markedly different from t hat found in fully differentiated fibers. Such diversity of this characteristic suggests t hat differentiation may commence at or before the time of birth. This observation is in keeping with the notion t hat diversification of fiber structure occurs in response to neural influence (11). Accordingly, fibers fail to differentiate histochemically in aneural muscle tissue culture (2) and lack ultrastructural specialization following fetal (32) or neonatal (49) denervation. From these findings it would appear t hat neural regulation is the major determinant of ultrastructural fiber diversification or at least facilitates the development

FIGS. 10-15. Variation in fiber ultrastructure during early p o s t n a t a l g r o w t h . All figures x 23 000. Fia. 10. Neonatal plantaris. An example of a fiber with relatively thick Z-line similar to those found in intermediate f b e r s . FIG. 11. Neonatal soleus. A fiber with relatively thin Z-lines is illustrated. Since the adult soleus is virtually homogeneous with regard to its fiber population (all fibers have thick Z-lines), differentiation presumably involves considerable growth in Z-line width in some fibers. FIG. 12. Soleus 3 weeks postnatal. This fiber typifies the soleus at this stage. Many of the characteristics of the intermediate fiber are seen; relatively wide Z-lines and a dense population of small (usually pleomorphic) mitochondria. Fro. 13. Plantaris 3 weeks postnatal.. At this age isolated triads (arrowheads) are numerous, although they usually lack orientation at the A-I band junction. FIG. 14. Plantaris 3 weeks postnatal. This fiber contains thin Z-lines (typical of white fibers) but has numerous, long mitochondria (typical of red fibers). A focus of filaments with associated small dense granules (ribosomes and polyribosomes) suggests the formation of a new myofibril (arrowheads) between adjacent myofibrils. FIG. 15. Plantaris 3 weeks postn&tal. A fiber with wide Z-band is contrasted with those shown in Figs. 13 and 14. FIGS. 16-18. Sarcomere formation in plantaris fibers. All figures x 23 000. FIa. 16. Z-lines of adjacent sarcomeres lie in close proximity and suggest that new sarcomeres may be formed to bridge the gaps between myofibrils (between arrowheads). The possibility of the gaps resulting from section orientation is ruled out since the adjacent Z-lines are in close proximity, i.e., only about onethird of a sarcomere. FIG. 17. Filaments are oriented but Z-lines (Z) are not well established. This mode of sarcomere formation was not as frequently observed as that shown in Fig. 18. Although the fibrils are contracted, the poorly developed Z-lines are evident. This appearance was more common in neonatal muscles. FIG. 18. A forming myofibril lies in the periphery of a fiber. The formation of the I-band (I) lags behind that of the A-band (A); the Z-line appears to develop in conjunction with the assembly of the thin filaments. Micrographs of cross-sectional fields support this hypothesis since most I-bands are considerably more narraw than A-bands. ".

220

221

222

ROBERT J. TOMANEK

of p h e n o t y p e in genetically p r e d e t e r m i n e d cells (32). Thus, both trophic factors (27) a n d the p a t t e r n of i m p u l s e a c t i v a t i o n (45) m a y be o p e r a t i o n a l at the t i m e of birth. The continued influence of n e u r a l factors as well as the role of muscle tension or stretch (30) p r e s u m a b l y contribute to the g r a d u a l process of differentiation d u r i n g e a r l y p o s t n a t a l growth.

Muscle Fiber Growth It is g e n e r a l l y a s s u m e d t h a t muscle fibers grow in d i a m e t e r as a r e s u l t of myofibril splitting (25), while l o n g i t u d i n a l g r o w t h is accomplished m a i n l y b y the form a t i o n of new s a r c o m e r e s at the end regions of the fiber (26, 59). O u r own observations indicate t h a t the proliferation of myofibrils occurs by two avenues: fiber splitting a n d t h e de novo f o r m a t i o n of m y ofibrils. The f o r m e r is s u g g e s t e d by the n u m e r o u s forked myofibrils which a r e of sufficient width, i.e., a p p r o x i m a t e l y one micron (25). De novo f o r m a t i o n is evident since n u m e r o u s foci of f i l a m e n t s occur in growing fibers; such f i l a m e n t s are a l w a y s associated w i t h ribosomes a n d polyribosomes. The conclusion t h a t n e w s a r c o m e r e s are added p r i m a r i l y n e a r the m y o t e n d o n junction is b a s e d on studies utilizing t r i t i a t e d adenosine which is incorporated into the s t r u c t u r a l A D P of actin f i l a m e n t s as well as into r i b o s o m a l R N A (26, 59). While our observations are in concert w i t h this conclusion, it should be e m p h a s i z e d t h a t this is not the sole m e a n s of l o n g i t u d i n a l

growth. At least in some fibers, t h e r e occur regions w i t h Z-line s t r e a m i n g a n d incomplete s a r c o m e r e s s u g g e s t i n g t h a t sarcomereogenesis m a y occur i n t e r n a l l y , i.e., e i t h e r b e t w e e n existing s a r c o m e r e s , or w h e r e myofibrils are in a s t a g e of formation at t h e i r t e r m i n a l portions possibly connecting two fibril s e g m e n t s . This interp r e t a t i o n does not contradict the studies utilizing labeled adenosine, since some label (although m i n i m a l ) occurs in the midbelly region of growing muscles.

Development of the Sarcotubular System The e a r l y p o s t n a t a l d e v e l o p m e n t of the t u b u l a r s y s t e m h a s b e e n e x t e n s i v e l y studied in the r a t (14, 48). In concert w i t h these studies, the p r e s e n t w o r k h a s demo n s t r a t e d t h a t the sarcoplasmic r e t i c u l u m , in the cat, u n d e r g o e s considerable developm e n t d u r i n g the first 2 to 3 w e e k s of postn a t a l life, a n d t h a t the n u m b e r of triads increases s u b s t a n t i a l l y d u r i n g this period. Similarly, increases in the v o l u m e of sarcoplasmic r e t i c u l u m d u r i n g the first 2 m o n t h s of e x t r a u t e r i n e life in b o t h fasta n d slow-twitch muscles h a s p r e v i o u s l y b e e n d e m o n s t r a t e d (36). The f o r m a t i o n of diads a n d t r i a d s app e a r s to be r e l a t e d to the r a p i d increases fil fiber d i a m e t e r b e t w e e n b i r t h a n d 2 w e e k s and is considered to comprise a coordin a t e d d e v e l o p m e n t , i.e., as fiber d i a m e t e r increases the T - t u b u l e s p e n e t r a t e deeper into the cell a n d m a k e contact w i t h compon e n t s of the s a r c o p l a s m i c r e t i c u l u m (23). O u r own o b s e r v a t i o n s a n d d a t a , however,

FIG. 19. Sarcomere formation in a soleus fiber. Z-line substance (Z) lies in the midst of disorganized contractile filaments. An assembled sarcomere is shown at the left of the field. This pattern of sarcomere and myofibril formation typifies fibers of the soleus, x 23 000. FIG. 20. Continuity of transverse tubules and sarcoplasmic reticulum (plantaris 6 weeks postnatal). As sarcomere and myofibril formation continue, the sarcoplasmic reticulum develops continuity between adjacent triads (arrowheads). x 23 000. FIG. 21. Plantaris 3 weeks compensatory hypertrophy. Disoriented components of sarcomeres include primarily the I-band region with streaming of the Z-lines (Z). Myofibrils are frequently split, x 23 000. FIo. 22. Plantaris 3 weeks compensatory hypertrophy. Some myofibrils (bottom of field) are oriented perpendicular to other myofibrils. Z, Z-line. x 23 000. Fro. 23. Plantaris 3 weeks compensatory hypertrophy. A myotube with numerous polyribosomes, mitochondria (mi), and peripheral myofibrils. Although not common to compensatory hypertrophy, myotubes are virtually nonexistent in fibers from 3-week-old controls.

SKELETAL MUSCLE FIBER DIVERSITY indicate some c h a n g e s in the s a r c o p l a s m i c r e t i c u l u m which h a v e not b e e n p r e v i o u s l y reported. The v o l u m e of this organelle differs in the A- a n d I - b a n d regions to a limited e x t e n t at b i r t h a n d increases in both regions d u r i n g the n e x t 3 weeks. This change is due m a i n l y to dilatation, r a t h e r t h a n to proliferation of the tubules. A considerable decrease in the r e l a t i v e v o l u m e of this organelle b e t w e e n 3 a n d 6 w e e k s s e e m s to be the r e s u l t of s u b s t a n t i a l increases in the v o l u m e of contractile filam e n t s a n d a r e t r a c t i o n of the dilated reticu l u m . The m a j o r increase in the r e l a t i v e volume of the r e t i c u l u m b e t w e e n 6 a n d 12 w e e k s is a r e s u l t of proliferation a n d gives rise to a h i g h l y organized a n d specialized a r r a n g e m e n t of the r e t i c u l a r n e t w o r k s s i m i l a r to t h a t p r e v i o u s l y described in various types of twitch fibers in the g u i n e a pig

(53). The p o s t n a t a l d e v e l o p m e n t of the sarcot u b u l a r s y s t e m a p p e a r s to correlate with changes in contractile properties. Differences b e t w e e n fast a n d slow muscles become m a r k e d a n d t h e a d u l t v a l u e s for velocity of s h o r t e n i n g (7) and m a x i m u m r a t e of tension d e v e l o p m e n t (8) a r e a t t a i n e d at the age of 6 weeks. I t is of i n t e r e s t t h a t our d a t a indicate a r e l a t i v e l y low v o l u m e of sarcoplasmic r e t i c u l u m at this age. However, the r e t i c u l u m is s u b s t a n t i a l l y specialized and u n d o u b t e d l y h a s a h i g h surface area; b u t e v e n m o r e significant, the r e t i c u l u m of fast- a n d slow-twitch fibers m a y differ w i t h respect to functional properties. For e x a m p l e , f r a g m e n t e d r e t i c u l u m for fast muscles h a s b e e n s h o w n to accum u l a t e Ca ~÷ at a m u c h f a s t e r r a t e t h a n

223

f r a g m e n t s isolated from slow-twitch m u s cle (21). According to this criteria, b o t h red and w h i t e fibers are considered to b e fast-twitch while i n t e r m e d i a t e fibers (soleus) are considered to be slow-twitch. Thus, both the e x t e n s i v e n e s s of the sarcoplasmic r e t i c u l u m and its c a l c i u m - b i n d i n g abilities a p p e a r to correlate w i t h velocity of shortening. As shown here, the v o l u m e and e x t e n s i v e n e s s of the r e t i c u l u m is g r e a t e s t in the w h i t e a n d lowest in the i n t e r m e d i a t e fiber. I f one also considers m y o s i n A T P a s e activity, which is r e l a t e d to velocity of s h o r t e n i n g (3), t h e n t h e r e a p p e a r s to be a strong a r g u m e n t for the contention t h a t b o t h red a n d w h i t e fibers, both of which s t a i n d a r k for m y o s i n A T P ase, are fast-twitch while i n t e r m e d i a t e fibers are slow-twitch (4). The observed increase in the n u m b e r of triads, t h e i r orie n t a t i o n at the A - I junction a n d the possible f o r m a t i o n of ~tight j u n c t i o n s , " which provide the link b e t w e e n the T t u b u l e s a n d the r e t i c u l u m (58), m a y also r e l a t e to postn a t a l c h a n g e s in contractile properties. In the r a t e x t e n s o r d i g i t o r u m longus a n d soleus muscles the e x c i t a t i o n - c o n t r a c t i o n latency period (the t i m e b e t w e e n the beginning of the action p o t e n t i a l a n d the onset of recorded d e v e l o p m e n t of tension) shortens m a r k e d l y d u r i n g the first 10 to 15 d a y s a f t e r b i r t h (10). In c o m p a r i s o n to fast-twitch muscles the fibers of the soleus h a v e a less e x t e n s i v e T - s y s t e m a n d such differences m a y contribute to the fact t h a t the soleus h a s a slower speed of contraction (36). A m i n o r i t y p o p u l a t i o n of fibers in the p l a n t a r i s is u l t r a s t r u c t u r a l l y s i m i l a r to

FIGS. 24-27. Ultrastructural characteristics of adult muscle fibers. All figures are × 23 000. FIG. 24. Red fiber (plantaris). Moderately wide Zdines and relatively large mitochondria (note the one in the extreme left of the field) typify this fiber type. FIG. 25. White fiber (plantaris). Such large fibers are typified by thin Z-lines and a sparsity of mitochondria (which are notably small). The sarcoplasmic reticulum is most extensive in this fiber type. FIG. 26. A fiber which is rarely found (soleus) has characteristics which do not enable its classification. Often small paired mitochondria occur at the Z-line; the latter is not as wide as that typifying intermediate fibers. FIG. 27. Intermediate fiber (soleus). This type of fiber typifies the soleus and is also found as a minority population in the heterogeneous plantaris. Z-lines are wide; mitochondria are usually small and paired at the Z-line. Lipid inclusions (li) are classically'numerous.

224

ROBERT J. TOMANEK

SKELETAL MUSCLE FIBER DIVERSITY the typical fiber found in soleus (tubular characteristics and volume, and Z-line dimensions) and is also light-staining for myosin ATPase (52). Such evidence, although without physiological correlates, suggests that these fibers may constitute a population of the slow-twitch variety.

Diversification of Fiber Types

225

virtually homogeneous fiber population at maturity a finding which is in concert with the muscles histochemical profile (52). Ultrastructural diversification of muscle fibers in the adult cat is similar to that observed in the guinea pig (18, 19, 53, 54). The Z-line data presented here approximate those reported by Eisenberg and colleagues (18, 19) who carried out extensive stereological analyses in guinea pig muscles. It is evident t h a t a number of fiber characteristics are autogenic, e.g., formation of triads, while those associated with specialization in structure and function are neurogenic, e.g., mitochondrial and sarcoplasmic reticulum content and distribution (32). The neural influence m a y be gradual as a process of motor neuron diversification precedes muscle fiber differentiation (44), a hypothesis which is consistent with the time gap between establishing motor end plates and muscle fiber diversification. Genetic factors similarly influence muscle fiber characteristics since ultrastructural characteristics of fast and slow muscle are not totally transformed by foreign innervation (32).

It is apparent that histochemical changes during the postnatal period are independent of each other and do not have an identical time course (52). Soleal fibers, for example, are always similar in SDH intensity regardless of their myosin ATPase activity. Such factors illustrate the complexity of the differentiation process as regards enzymatic and structural characteristics. The time course of ultrastructural differentiation appears to vary for the three fiber types. Fibers with characteristics of the intermediate type can be found during the first few weeks following birth while fibers with characteristics of the white type are not clearly differentiated until the period of 6 to 10 weeks. It has been postulated that three classes of fiber types develop as three distinct populations (60). In general, histochemical data fail to support Compensatory Hypertrophy Functional overload of undifferentiated this hypothesis (52). However, in spite of early similarities in myosin ATPase and fibers results in marked hypertrophy of all oxidative enzyme activities, and subse- fiber types and to some extent affects the quent alterations in these activities in cer- histochemical profile in slow- and fasttain fibers, there occurs considerable vari- twitch muscles (52). Enhanced oxidative ation in Z-line widths and a relative varia- capacity as indicated by increased SDH tion in sarcoplasmic reticulum. Since the activity is common to functional overload various histochemical and ultrastructural resulting from extirpation of a synergist characteristics show a relative independ- (46, 52) as well as exercise training (5, 20, ence during developmental stages, a true 35). Since SDH activity is correlated with and complete conversion of fiber type does resistance to fatigue (9, 17) its enhancenot occur during differentiation. Rather, ment after functional overload represents changes in specific characteristics contrib- an important adaptation. The presence of ute to the differentiation process, which in a fairly abundant mitochondrial complethe heterogeneous plantaris consist of ac- m e n t in the hypertrophic plantaris (at 6 centuation of the diversity of ultrastruc- weeks) in fibers with relatively narrow Ztural characteristics. In the soleus the fi- lines~suggests a specific modification of the ber population shows-less ultrastructural classical white fiber, stimulated by the imdiversity with maturation and results in a posed tension and stretch (29).

226

ROBERT J. TOMANEK

The fact that Z-line width diversification in the hypertrophic plantaris muscle is similar to that of the controls suggests that this parameter is related to neural and/or genetic rather than peripheral factors. Whether the sarcoplasmic reticulum in red fibers is actually increased after functional overload is not conclusive. While the data indicate significant volume increases in this organelle, dilatation may, in part, account for such a change. In addition, the adaptive response of white fibers involving numerical increases in mitochondria makes phenotypic distinction of red and white fibers difficult in micrographs of cross-sectional fields. Thus some of the reticulum data from the hypertrophic plantaris muscles may have included fibers which were genetically of the white type. Increases in the extent of the sarcoplasmic reticulum are not consistent with the finding that the velocity of contraction in fast-twitch hypertrophic muscle is initially prolonged and later returns to normal (30, 57). Moreover, the volume of sarcoplasmic reticulum of fibers comprising the slow-twitch soleus is similar in hypertrophic and control specimens. Enhanced phasic activity of a muscle (e.g., swimming) does not necessarily lead to hypertrophy and has been shown to shorten contraction time (28). The major ultrastructural alteration in all fiber types of hypertrophic muscles involves an enhancement of the proliferation of myofibrils via splitting and de novo synthesis. In some fibers this enhancement includes the appearance of disoriented myofibrils similar to those seen during restitution of the fiber following atrophy (54). Increases in cell size during hypertrophy of this type appear to involve proportional increases in soluble and myofibrillar proteins (24). While total myosin is increased in hypertrophic muscles, it is structurally and enzymatically identical to that found in control muscles (33), a finding which suggests that this protein is genetically or neurally regulated.

An increase in the total number of muscle fibers after functional overload during the early postnatal period has been suggested (46). Satellite cells have been shown to increase substantially during the first week of surgically induced functional overload (47), but their fate remains to be determined. Their decrease after the fifth postoperative day may be due to their incorporation into muscle fibers which constitutes a normal process during postnatal growth (37). Alternately, fusion of satellite cells may give rise to new fibers. In concert with this possibility we occasionally found myotubes in hypertrophic muscles. Yet their occurrence was inconsistent and undoubtedly limited; thus, hyperplasia involving precursor cells is probably not a major factor in compensatory hypertrophy. On the other hand, splitting of fibers has been previously reported (31, 56) and would appear to be a more likely method of fiber proliferation. It has been noted that if an adequate stretch is maintained in the chicken anterior latissimus dorsi, large numbers of new fibers are produced (50). These authors have concluded that both hyperplasia and hypertrophy result from such a stimulus, although all of the new fibers presumably do not reach maturity. Undoubtedly the question of hyperplasia has not been adequately answered with respect to functional overload. In conclusion, functional overload during early postnatal growth affects those specific ultrastructural and histochemical properties of muscle fibers which enable the fiber to meet increased work demands. Such evidence together with the finding that other characteristics (e.g., Z-line width, myosin ATPase staining) are not significantly altered by functional overload, indicates the specificity of factors regulating muscle characteristics. REFERENCES 1. ASHMORE, C. R., TOMPKINS, G., AND DOERR, L., J. A n i m . Sci. 34, 37 (1972). 2. ASKANSAS, V., SHAFIQ, S. A., AND MILHORAT, A. T., Exp. Neurol. 37, 218 (1972).

SKELETAL MUSCLE FIBER DIVERSITY

227

248, 1056 (1973). 3. BARANY, M., J. Gen. Physiol. 50, 197 (1967). 4. BARNARD, R. J., EDGERTON, V. R., FURUKAWA, 34. KARPATI, G., AND ENGEL, W. K., Arch. Neurol. 17, 542 (1967). T., AND PETER, J. S., Amer. J. Physiol. 220, 35. KOWALSKI, K., GORDON, E. E., MARTINEZ, A., 410 (1971). AND ADANEK, J., J. Histochem. Cytochem. 17, 5. BARNARD,R. J., EDGERTON, V. R., AND PETER, J. 601 (1969). B., J. Appl. Physiol. 28, 762 (1970). 36. LUFF, A. R., AND ATWOOD, H. L., J. Cell Biol. 51, 6. BROOKE, M. H., AND KAISER, K. K., Arch. Neu369 (1971). rol. 23, 369 (1970). 7. BULLER, A. J., ECCLES, J. C., AND ECCLES, R~ 37. Moss, F. P., AND LEBLOND, C. P., Anat. Rec. 170, 421 (1971). M., J. Physiol. 150, 399 (1970). 38. NYSTROM, B., Acta Neurol. Scand. 44, 405 8. BULLER, A. J., AND LEWIS, D. M., J. Physiol. (1968). 176, 355 (1965). 39. OGATA, T., Acta Med. Okayana 18, 271 (1964). 9. BURKE, R. E., LEVINE, D. N., ZAJAC, F. E., TSAIRIS, P., AND ENGEL, W. K., Science 174, 40. OGATA, T., AND MURATA, F., Tohoku J. Exp. Med. 99, 225 (1969). 709 (1971). 41. PADYKULA,H. A., AND GAUTHIER, G. F., in MIL1O. CHAPLIN, E. R., NELL, G. W., AND WALKER, S. HORAT, A. T. (Ed.) Exploratory Concepts in M., Exp. Neurol. 29, 142 (1970). Muscular Dystrophy and Related Disorders, 11. CLOSE, R., J. Physiol. 173, 74 (1964). pp. 117-128. Excerpta Medica Foundation, 12. COOPER, C. C., CASSENS, R. G., KASTENSCHMIDT, Amsterdam, 1967. L. L., AND BRISKEY, E. J., Develop. Biol. 23, 42. PELLEGRINO, C., AND FRANZINI, C., J. Cell Biol. 169 (1970). 17, 327 (1963). 13. DUBOWITZ, V., Developing and Diseased Muscle: A Histochemical Study. Heinemann, Lon- 43. PETER, J. B., BARNARD, R. J., EDGERTON, V. R., GILLESPIE, C. A., AND STEMPEL, K. E., Biodon, 1968. chemistry 11, 2627 (1972). 14. EDGE, M. S., Develop. Biol. 23, 634 (1970). 15. EDGERTON, V. R., GERCHMAN, L., AND CARROW, 44. RIDGE, R. M., Quart. J. Exp. Physiol. 52, 293 (1967). R., Exp. Neurol. 24, 110 (1969). 45. RILEY, D. A., AND ALLIN, E. F., Exp. Neurol. 40, 16. EDGERTON, V. R., AND SIMPSON, D. R., J. Histo391 (1973). chem. Cytochem. 17, (1969). 46. SCHIAFFINO, S., AND BORMIOLI, S. P., Exp. Neu17. EDSTROM, L., AND KUGELBERG, E., J. Neurol. rol. 40, 126 (1973). Neurosurg. Psychiat. 31, 424 (1968). 47. SCHIAFFINO,S., BORMIOLI,S. P., AND ALOISI, M., 18. EISENBERG, B. B., KUDA, A. M., AND PETER, J. Virchow. Arch. (Zellpath) 11, 268 (1972). B., J. Cell Biol. 60, 732 (1974). 48. SCHIAFFINO,S., AND MARGRETH, A., J. Cell Biol. 19. EISENBERG, B. R., AND KUDA, A. M., J. Ultra41, 855 (1969). struct. Res., 51, 176 (1975). 49. SCHAFIQ, S. A., SIEDU, S. A., AND MILHORAT, A. 20. FAULKNER, J. A., MAXWELL, L. C., BROOK, D. T., Exp. Neurol. 35, 529 (1972). A., AND LIEBERMAN, D. A., Amer. J. Physiol. 50. SOLA, 0. M., CHRISTENSEN, D. L., AND MARTIN, 221, 291 (1971). A. W., Exp. Neurol. 41, 76 (1973). 21. FIEHN, W., AND PETER, J. B., J. Clin. Invest. 50, 51. STEIN, J. M., AND PADYKULA, H. A., Amer. J. 57O (1971). Anat. 110, 103 (1962). 22. FINOL, K. H., Tsitologiya 9, 347 (1967). 52. TOMANEK,R. J., Develop. Biol. 42,305 (1975). 23. GAUTHIER, G. F., AND PADYKULA,H. A., J. Cell 53. TOMANEK,R. J., ASMUNDSON,C. R., COOPER, R. Biol. 28, 333 (1966). R., AND BARNARD, R. J., J. Morph. 139, 47 24. GOLDBERG, A. L., J. Cell Biol. 36, 653 (1968). (1973). 25. GOLDSPINK, G., J. Cell Sci. 6, 593 (1970). 26. GRIFFIN, G. E., WILLIAMS, P., AND GOLDSPINK, 54. TOMANEK, R. J., AND COOPER, R. R., J. Anat. 113, 409 (1972). G., Nature (London) 232, 28 (1971). 55. TRAYER, I. P., AND PERRY, S. V., Biochem. Z. 27. GUTH, L., Physiol. Rev. 48, 654 (1968). 345, 78 (1966). 28. GUTMANN, E., AND HAJEK, I., Physiol. Bohe56. VAN LINGE, B., J. Bone Joint Surg. (Brit.) 44, moslov. 20, 205 (1971). 711 (1962). 29. GUTMANN, E., MELICHNA, J., AND SYROV~, I., 57. VRBOVA, G., J. Physiol. 169, 513 (1963). Experientia 29, 435 (1973). 30. GUTMANN, E., SCHIAFFINO, S., AND HANZLI- 58. WALKER,S. M., AND SCHRODT, G. R . , A n a t . Rec. 155, 1 (1966). KOVA, V., Exp. Neurol. 31, 451 (1971). 59. WILLIAMS,P. E., AND GOLDSPINK;G., J. Cell Sci. 31. HALL-CRAGGS,E. C. B., J. Anat. 107, 459 (1970). 9, 751 (1971). 32. HANZLIKOVA, V., AND SCHIAFFINO, S., Z. Zell60. WIRSEN, C., AND LARSSON, K. S., J. Embryol. forsch. 147, 75 (1973). Exp. Morph.'12, 759 (1964). 33. JABLECKI, C., AND KAUFMAN, S., J. Biol. Chem.

Ultrastructural differentiation of skeletal muscle fibers and their diversity.

JOURNAL OF U L T R A S T R U C T U R E RESEARCH 55, 212-227 (1976) Ultrastructural Differentiation of Skeletal Muscle Fibers and Thei~ Diversity...
14MB Sizes 0 Downloads 0 Views