Ribosomes in the skeletal muscle filament lattice.

The compartmentalization of myosin isoforms within a muscle cell (Gauthier: J. Cell Biol. 110:693-701, 1990) suggests that myosin might be assembled directly into thick filaments at sites where it is synthesized. We therefore examined myofibrils by immunoelectron microscopy to determine whether ribosomes are associated with thick filaments under conditions in which new myosin can be identified. We used the embryonic chick anterior latissimus dorsi (ALD), a slow muscle that is induced, by curare, to synthesize a fast myosin isoform that is not normally present. Myosin was localized in situ, using a gold-labeled monoclonal antibody that recognizes the new isoform. The gold marker, as expected, was localized preferentially to the A band. There was an overall increase of fivefold in the number of gold particles per micron2 of A band in the curare-treated compared to the normal ALD, indicating that the labeled isoform was largely newly formed. There was a corresponding preferential distribution of ribosomes at the A band, especially in the H-band region, and the number of ribosomes per micron2 of A band was nearly twice as high in the curare-treated as in the normal muscle. Ribosomes were located between thick filaments, often aligned in rows. We conclude that ribosomes are located within the filament lattice, and therefore that they are available for local myosin synthesis.

Developmental transitions in the myosin patterns of two fast muscles.

Transitions in myosin patterns were examined in situ by immunofluorescence in two fast muscles of the developing chicken, the pectoralis and the posterior latissimus dorsi. Myosin isoforms were localized using stage-specific monoclonal antibodies against the heavy chain of pectoralis myosin. Two antibodies (12C5 and 10H10) recognize adult and late embryonic myosin. They reacted weakly with both the pectoralis and posterior latissimus dorsi at 10 days in ovo, but intensely at 18 days in ovo. Both muscles were completely unreactive with an adult-specific antibody (5C3), indicating that the staining with 12C5 and 10H10 at 18 days in ovo reflects embryonic myosin. Thus two different embryonic isoforms are expressed sequentially in each muscle. Both 12C5 and 10H10 reacted weakly again with these muscles after hatching. The reappearance of a strong positive response to both antibodies, at 28 days in the pectoralis and after 60 days in the posterior latissimus dorsi, correlated well with the first appearance of a response to the adult-specific antibody, 5C3, signalling the beginning of the adult pattern. Both muscles reacted strongly with an antibody (5B4) specific for 'neonatal' myosin between 18 days in ovo and 60 days after hatching. In the pectoralis, embryonic was replaced by neonatal myosin in most fibres by 14 days after hatching; by 28 days, both adult and neonatal myosin were expressed in most fibres; and in the adult, neonatal myosin was replaced entirely by the adult isoform. In contrast, many fibres in the posterior latissimus dorsi still expressed both embryonic and neonatal myosins up to at least 60 days post-hatch, and the remaining fibres expressed the neonatal isoform; the neonatal isoform was present in some fibres even in the adult posterior latissimus dorsi. We have therefore demonstrated in situ four different heavy chain isoforms in two different fast muscles. 'Early embryonic', 'late embryonic', 'neonatal' and eventually 'adult' isoforms are expressed in each muscle and more than one isoform often coexists in the same fibre.

Differential distribution of myosin isoforms among the myofibrils of individual developing muscle fibers.

Myosin was localized in situ in the posthatch chicken pectoralis using isoform-specific mAbs. The distribution among myofibrils was demonstrated by immunofluorescence and by immunogold EM. Fluorescein- or rhodamine-labeled antibody (12C5) specific for the head region (S1) of myosin was used as a marker to identify "embryonic" myosin. In longitudinal semithin frozen sections, a minority population of myofibrils stained intensely with 12C5. All other myofibrils in the same cell stained only weakly. Similarly, in Lowicryl-embedded ultrathin sections prepared for EM, a minority population reacted preferentially with gold-labeled 12C5. An antibody (5B4) specific for the rod portion of "neonatal" myosin reacted strongly with nearly all myofibrils, and this was evident by light and electron microscopy. A few of the fibrils that reacted strongly with 12C5 reacted weakly with 5B4. These observations demonstrate that an epitope reacting with 12C5 is more abundant in some myofibrils than in others within the same cell. Three categories of myofibrils can be identified by their relative proportions of embryonic and neonatal forms of myosin: in nearly all fibrils, a neonatal isoform predominates; in a minority population, embryonic and neonatal isoforms are both abundant; and in a few fibrils, an embryonic isoform predominates. It is concluded that there are distinct populations of myofibrils in which specific isoforms are segregated within an individual cell.

Neuromuscular junctions in adult and developing fast and slow muscles.

Functional changes that occur just before hatching in future fast muscles of the chicken are thought to be influenced by the pattern of innervation. We have compared the neuromuscular junctions of two fast muscles, the posterior latissimus dorsi (PLD) and the pectoralis, which differ in their myosin composition at 18 days in ovo. We have also presented new information on the neuromuscular junctions of the adult fast muscles and an adult slow muscle, the anterior latissimus dorsi (ALD). Both categories of adult muscles were heterogeneous, and there was little difference between endplates of the two fast muscles or between the fast and slow muscles. In contrast, there were significant structural differences between the two fast muscles during embryonic development. In early embryonic muscle fibers, which synthesize embryonic forms of myosin, individual motor endplates were contacted by multiple axon terminals. At 18 days in ovo, the majority of the neuromuscular junctions in the pectoralis continued to be multiterminal, whereas all but one of the terminals had been withdrawn from each endplate in the PLD. This single terminal had a unique form that distinguished it from the embryonic pectoralis and also from the two adult muscles. By 7 days after hatching, the neuromuscular junctions of both muscles had single terminals. They were different from the embryonic terminals, though not necessarily equivalent to adult terminals. The results show that multiple terminals persist at 18 days in ovo in the muscle that continues to express an embryonic myosin, but they have been withdrawn from the muscle that has lost this myosin. It is concluded, from combined data on the two muscles, that maturation of the neuromuscular junction during embryonic and late posthatch development is correlated with transitions in the myosin pattern and in contractile properties.

Freeze-fractured sarcoplasmic reticulum in adult and embryonic fast and slow muscles.

There is evidence to suggest that 8 nm calcium transport particles in the sarcoplasmic reticulum are involved in the regulation of twitch properties in adult muscles. We have studied ultrastructural characteristics of the sarcoplasmic reticulum in relation to previously defined physiological changes that take place in the normal course of development. The fast twitch posterior latissimus dorsi (PLD) and the slow tonic anterior latissimus dorsi (ALD) of the chicken were compared using the procedure of freeze-fracture. In the adult PLD, the sarcoplasmic reticulum was composed of longitudinal tubules which gave rise to fenestrated cisternae at the centre of the H band and to terminal cisternae that form triads regularly at each A-I junction. In most of the fibres (85%), 8 nm intramembrane particles were closely packed in the concave fracture face (P-face). In the ALD, a tubular network with an open circular pattern extended the entire length of the A band and usually throughout the I band as well. Dyads or triads, which were infrequent, were often oriented obliquely. The density of intramembrane particles was low in the majority of the fibres, but there was a significant minority population (30%) in which particle density was relatively high. At 10 days in ovo, when speed of contraction in both the ALD and PLD is slow, there was a circular configuration of sarcoplasmic reticulum components in both muscles, and particle density was low. Surprisingly, at 18 days in ovo, when the rate of tension development and relaxation have reached nearly adult values in the fast PLD, this muscle, like the ALD, continued to exhibit a circular arrangement of sarcoplasmic reticulum tubules. The density of P-face particles, although greater than at 10 days, was still low relative to the adult PLD. Estimated values for the 18-day PLD were similar to those calculated for the adult slow muscle. Our observations, along with those of other investigators, suggest that abundant intramembrane particles may be related to the fast twitch properties of the adult PLD. However, they indicate that neither the pattern of membranes typical of the adult fast muscle nor the high content of calcium transport particles is required for the differentiation of fast twitch characteristics.

Curare-induced transformation of myosin pattern in developing skeletal muscle fibers.

The effects of neuromuscular block on the pattern of distribution of myosin isozymes in developing skeletal muscle fibers was examined by immunocytochemistry. The homogeneous population of fibers in the anterior latissimus dorsi (ALD) of the 18-day chick embryo was converted by curare to a mosaic of at least two categories of fibers. Normally all fibers in this slow muscle reacted with antibodies against slow myosin (anti-ALD). They also reacted with an antibody specific for the alkali 1 light chain (anti-delta 1) but not the alkali 2 light chain (anti-delta 2) of fast myosin. After treatment with curare, which inhibits neuronal cell death and increases the number of axonal endings, ALD muscle fibers continued to react with anti-delta 1, but many now reacted with anti-delta 2 as well. The same fibers failed to react with anti-ALD. From this it can be concluded that the myosin in this population was converted to a type not normally present. The changes, therefore, are not merely a result of the preferential loss of a slow type of fiber, nor are they a result of delayed maturation. In contrast, curare had no apparent effect on the fast posterior latissimus dorsi (PLD). As in the normal muscle at 18 days, all fibers reacted strongly with anti-delta 1 and to variable degrees with anti-delta 2, and very few fibers reacted with anti-ALD. Our observations suggest that the dual response to antibodies against fast and slow myosin during development is not a necessary consequence of multiple axon terminals. We present evidence that curare induces the expression of a different myosin in the embryonic ALD, and we suggest that the selective transformation of the fiber population may be a manifestation of a change in composition of the motoneuron pool.

Myosin isozymes in normal and cross-reinnervated cat skeletal muscle fibers.

Immunocytochemical characteristics of myosin have been demonstrated directly in normal and cross-reinnervated skeletal muscle fibers whose physiological properties have been defined. Fibers belonging to individual motor units were identified by the glycogen-depletion method, which permits correlation of cytochemical and physiological data on the same fibers. The normal flexor digitorum longus (FDL) of the cat is composed primarily of fast-twitch motor units having muscle fibers with high myosin ATPase activity. These fibers reacted with antibodies specific for the two light chains characteristic of fast myosin, but not with antibodies against slow myosin. Two categories of fast fibers, corresponding to two physiological motor unit types (FF and FR), differed in their immunochemical response, from which it can be concluded that their myosins are distinctive. The soleus (SOL) consists almost entirely of slow-twitch motor units having muscle fibers with low myosin ATPase activity. These fibers reacted with antibodies against slow myosin, but not with antibodies specific for fast myosin. When the FDL muscle was cross-reinnervated by the SOL nerve, twitch contraction times were slowed about twofold, and motor units resembled SOL units in a number of physiological properties. The corresponding muscle fibers had low ATPase activity, and they reacted with antibodies against slow myosin only. The myosin of individual cross-reinnervated FDL muscle units was therefore transformed, apparently completely, to a slow type. In contrast, cross-reinnervation of the SOL muscle by FDL motoneurons did not effect a complete converse transformation. Although cross-reinnervated SOL motor units had faster than normal twitch contraction times (about twofold), other physiological properties characteristic of type S motor units were unchanged. Despite the change in contraction times, cross-reinnervated SOL muscle fibers exhibited no change in ATPase activity. They also continued to react with antibodies against slow myosin, but in contrast to the normal SOL, they now showed a positive response to an antibody specific for one of the light chains of fast myosin. The myosins of both fast and slow muscles were thus converted by cross-reinnervation, but in the SOL, the newly synthesized myosin was not equivalent to that normally present in either the FDL or SOL. This suggests that, in the SOL, alteration of the nerve supply and the associated dynamic activity pattern are not sufficient to completely respecify the type of myosin expressed.

Distribution and properties of myosin isozymes in developing avian and mammalian skeletal muscle fibers.

Isozymes of myosin have been localized with respect to individual fibers in differentiating skeletal muscles of the rat and chicken using immunocytochemistry. The myosin light chain pattern has been analyzed in the same muscles by two-dimensional PAGE. In the muscles of both species, the response to antibodies against fast and slow adult myosin is consistent with the speed of contraction of the muscle. During early development, when speed of contraction is slow in future fast and slow muscles, all the fibers react strongly with anti-slow as well as with anti-fast myosin. As adult contractile properties are acquired, the fibers react with antibodies specific for either fast or slow myosin, but few fibers react with both antibodies. The myosin light chain pattern slow shows a change with development: the initial light chains (LC) are principally of the fast type, LC1(f), and LC2(f), independent of whether the embryonic muscle is destined to become a fast or a slow muscle in the adult. The LC3(f), light chain does not appear in significant amounts until after birth, in agreement with earlier reports. The predominance of fast light chains during early stages of development is especially evident in the rat soleus and chicken ALD, both slow muscles, in which LC1(f), is gradually replaced by the slow light chain, LC1(s), as development proceeds. Other features of the light chain pattern include an "embryonic" light chain in fetal and neonatal muscles of the rat, as originally demonstrated by R.G. Whalen, G.S. Butler- Browne, and F. Gros. (1978. J. Mol. Biol. 126:415-431.); and the presence of approximately 10 percent slow light chains in embryonic pectoralis, a fast white muscle in the adult chicken. The response of differentiating muscle fibers to anti-slow myosin antibody cannot, however, be ascribed solely to the presence of slow light chains, since antibody specific for the slow heavy chain continues to react with all the fibers. We conclude that during early development, the myosin consists of a population of molecules in which the heavy chain can be associated with a fast, slow, or embryonic light chain. Biochemical analysis has shown that this embryonic heavy chain (or chains) is distinct from adult fast or slow myosin (R.G. Whalen, K. Schwartz, P. Bouveret, S.M. Sell, and F. Gros. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:5197-5201. J.I. Rushbrook, and A. Stracher. 1979. Proc Natl. Acad. Sci. U.S.A. 76:4331-4334. P.A. Benfield, S. Lowey, and D.D. LeBlanc. 1981. Biophys. J. 33(2, Pt. 2):243a[Abstr.]). Embryonic myosin, therefore, constitutes a unique class of molecules, whose synthesis ceases before the muscle differentiates into an adult pattern of fiber types.

Ultrastructural identification of muscle fiber types by immunocytochemistry.

In a fast-twitch muscle, three types of fibers (red, intermediate, and white) can be distinguished on the basis of mitochondrial content. Red fibers, identified by abundant mitochondria, can be further differentiated on the basis of a positive or negative response to antibodies specific for white ("fast") myosin. Because there is also a difference in Z-line width among fibers of the same muscle, the possibility existed that the two red fibers, which differ in type of myosin, might also differ in dimensions of the Z line. We therefore examined, with the electron microscope, fibers which had been exposed to antibody against white myosin. In those fibers which react with the antibody, an electron-opaque band is evident in the H-band region, thereby distinguishing reactive from unreactive fibers. The red fiber can now be subdivided on the basis of a positive or negative response to anti-white myosin visualized directly with the electron microscope. Both categories of red fibers ("fast" and "slow") have wide Z lines, and thus are distinguished from white and intermediate fibers, which react with the antibody but which have narrow Z lines. On the basis of combined immunocytochemical and ultrastructural characteristics, four types of fibers can be recognized in a single muscle. Moreover, it is demonstrated here that a wide Z line does not necessarily imply a slow speed of contraction.

Distribution of myosin isoenzymes among skeletal muscle fiber types.

Using an immunocytochemical approach, we have demonstrated a preferential distribution of myosin isoenzymes with respect to the pattern of fiber types in skeletal muscles of the rat. In an earlier study, we had shown that fluorescein-labeled antibody against "white" myosin from the chicken pectoralis stained all the white, intermediate and about half the red fibers of the rat diaphragm, a fast-twitch muscle (Gauthier and Lowey, 1977). We have now extended this study to include antibodies prepared against the "head" (S1) and "rod" portions of myosin, as well as the alkali- and 5,5'dithiobis (2-nitrobenzoic acid) (DTNB)-light chains. Antibodies capable of distinguishing between alkali 1 and alkali 2 type myosin were also used to localize these isoenzymes in the same fast muscle. We observed, by both direct and indirect immunofluorescence, that the same fibers which had reacted previously with antibodies against white myosin reacted with antibodies to the proteolytic subfragments and to the low molecular-weight subunits of myosin. These results confirm our earlier conclusion that the myosins of the reactive fibers in rat skeletal muscle are sufficiently similar to share antigenic determinants. The homology, furthermore, is not confined to a limited region of the myosin molecule, but includes the head and rod portions and all classes of light chains. Despite the similarities, some differences exist in the protein compositions of these fibers: antibodies to S1 did not stain the reactive (fast) red fiber as strongly as they did the white and intermediate fibers. Non-uniform staining was also observed with antibodies specific for A2 myosin; the fast red fiber again showed weaker fluorescence than did the other reactive fibers. These results could indicate a variable distribution of myosin isoenzymes according to their alkali-light chain composition among fiber types. Alternatively, there may exist yet another myosin isoenzyme which is localized in the fast red fiber. Those red fibers which did not react with any of the antibodies to pectoralis myosin, did react strongly with an antibody against myosin isolated from the anterior latissimus dorsi (ALD), a slow red muscle of the chicken. The myosin in these fibers (slow red fibers) is, therefore, distinct from the other myosin isoenzymes. In the rat soleus, a slow-twitch muscle, the majority of the fibers reacted only with antibody against ALD myosin. A minority, however, reacted with antiboddies to pectoralis as well as ALD myosin, which indicates that both fast and slow myosin can coexist within the same fiber of a normal adult muscle. These immunocytochemical studies have emphasized that a wide range of isoenzymes may contribute to the characteristic physiological properties of individual fiber types in a mixed muscle.

Fast and slow myosin in developing muscle fibres.

Slow and fast isoenzymes of myosin coexist in all the fibres of a fast-twitch mammalian muscle during early development. They later become segregated into different populations of fibres. Slow myosin is most abundant when the speed of contraction of the muscle is slow and the fibres are multiply innervated; its synthesis in the majority of the fibres seems to be 'switched off' when the speed of contraction increases and the fibres become innervated by single motoneurones.

Polymorphism of myosin among skeletal muscle fiber types.

An immunocytochemical approach was used to localize myosin with respect to individual fibers in rat skeletal muscle. Transverse cryostat sections of rat diaphragm, a fast-twitch muscle, were exposed to fluorescein-labeled immunoglobulin against purified chicken pectoralis myosin. Fluorescence microscopy revealed a differential response among fiber types, identified on the basis of mitochondrial content. All white and intermediate fiber but only about half of the red fiber reacted with his antimyosin. In addition, an alkali-stable ATPase had the same pattern of distribution among fibers, which is consistent with the existence of two categories of red fibers. The positive response of certain red fibers indicates either that their myosin has antigenic determinants in common with "white" myosin, or that the immunogen contained a "red" myosin. Myosin, extracted from a small region of the pectorlis which consists entirely of white fibers, was used to prepare an immunoadsorbent column to isolate antibodies specific for white myosin. This purified anti-white myosin reacted with the same fibers of the rat diaphragm that had reacted with the white, intermediate, and some red fibers are sufficiently homologous to share antigenic determinants. In a slow-twitch muscle, the soleus, only a minority of the fiber reacted with antipectoralis myosin. The majority failed to respond; hence, they are not equivalent to intermediate fibers of the diaphragm; despite their intermediate mitochondrial content. Immunocytochemical analysis of two different musles of the rat has demonstrated that more than one isoenzyme of myosin can exist in a single muscle, and that individual fiber types can be recognized by immunological differences in their myosin. We conclude that, in the rat diaphragm, there are at least two immunochemically distinct types of myosin and four types of muscle fibers: white, intermediate, and two red. We suggest that these fibers correspond to the four types of motor units described by Burke et al. (Burke, R. E., D. N. Levine, P. Tsairis, and F. E. Zajac, III 1973. J. Physiol. (Lond) 234:723-748.)in the cat gastrocnemius.;

The ultrastructure of the neuromuscular junctions of mammalian red, white, and intermediate skeletal muscle fibers.

Distinct ultrastructural differences exist at the neuromuscular junctions of red, white, and intermediate fibers of a mammalian twitch skeletal muscle (albino rat diaphragm). The primary criteria for recognizing the three fiber types are differences in fiber diameter, mitochondrial content, and width of the Z line. In the red fiber the neuromuscular relationship presents the least sarcoplasmic and axoplasmic surface at each contact. Points of contact are relatively discrete and separate, and axonal terminals are small and elliptical. The junctional folds are relatively shallow, sparse, and irregular in arrangement. Axoplasmic vesicles are moderate in number, and sarcoplasmic vesicles are sparse. In the white fiber long, flat axonal terminals present considerable axoplasmic surface. Vast sarcoplasmic surface area is created by long, branching, closely spaced junctional folds that may merge with folds at adjacent contacts to occupy a more continuous and widespread area. Axoplasmic and sarcoplasmic vesicles are numerous. Both axoplasmic and sarcoplasmic mitochondria of the white fiber usually contain intramitochondrial granules. The intermediate fiber has large axonal terminals that are associated with the most widely spaced and deepest junctional folds. In all three fiber types, the junctional sarcoplasm is rich in free ribosomes, cisternae of granular endoplasmic reticulum, and randomly distributed microtubules.