Muscle fiber pattern is independent of cell lineage in postnatal rodent development.

Muscle fibers specialized for fast or slow contraction are arrayed in characteristic patterns within developing limbs. Clones of myoblasts analyzed in vitro express fast and slow myosin isoforms typical of the muscle from which they derive. As a result, it has been suggested that distinct myoblast lineages generate and maintain muscle fiber pattern. We tested this hypothesis in vivo by using a retrovirus to label myoblasts genetically so that the fate of individual clones could be monitored. Both myoblast clones labeled in muscle in situ and clones labeled in tissue culture and then injected into various muscles contribute progeny to all fiber types encountered. Thus, extrinsic signals override the intrinsic commitment of myoblast nuclei to particular programs of gene expression. We conclude that in postnatal development, pattern is not dictated by myoblast lineage.     

Shortening velocity and myosin heavy chains of developing rabbit muscle fibers

The regulation of vertebrate muscle contraction with respect to the role of the different subunits of myosin remains somewhat uncertain. One approach to gaining a better understanding of the molecular basis of contraction is to study developing muscle which undergoes changes in myosin isozyme composition and contractile properties during the normal course of maturation. The present study utilizes single fibers from psoas muscles of rabbits at several ages as a model system for fast-twitch muscle development. This approach eliminates the inherent problems of interpreting results from studies on whole muscles which usually contain heterogeneous fiber types with respect to contractile properties and isoenzyme composition. Maximum velocity of shortening and tension-generating ability of individual fibers were measured and the myosin heavy chain composition of the same fibers was examined using an ultrasensitive sodium dodecyl sulfate-polyacrylamide gel system. The results indicate that 1) with regard to contractile properties, there is a transitional period from slow to fast shortening velocities within the first postnatal month; 2) a strong, positive correlation exists between the speed of shortening and tension-generating ability of individual postnatal day 7 fibers, suggesting that as more myosin is incorporated in these developing fibers it is of the fast type; and 3) there is a wide variation in maximum velocity of shortening among postnatal day 7 psoas fibers which is also a time when a mixture of heavy chain isoforms characterizes the myosin composition of single muscle fibers.     

Myosin types in human skeletal muscle fibers

By combining enzyme histochemistry for fiber typing with immunohistochemistry for slow and fast myosin a correlation between fiber type and myosin type was sought in human skeletal muscle. Fiber typing was done by staining for myofibrillar ATPases after preincubation at discriminating pH values. Myosin types were discriminated using type specific anti-rabbit myosin antibodies shown to cross-react with human myosin and were visualized by a protein A-peroxidase method. Type I fibers were shown to contain slow myosin only, type IIA and IIB fibers fast myosin only, and type IIC fibers both myosins in various proportions. When muscle biopsies from well-trained athletes were investigated essentially the same staining pattern was observed. However, rarely occurring type I fibers with high glycolytic activity were detected containing additional small amounts of fast myosin and occasional type IIA fibers had small amounts of slow myosin. Based on the observation of various fiber types in which slow and fast myosin coexist we propose a dynamic continuum of fibers encompassing all fiber types.     

Myosin types in human skeletal muscle fibers

Summary By combining enzyme histochemistry for fiber typing with immunohistochemistry for slow and fast myosin a correlation between fiber type and myosin type was sought in human skeletal muscle. Fiber typing was done by staining for myofibrillar ATPases after preincubation at discriminating pH values. Myosin types were discriminated using type specific anti-rabbit myosin antibodies shown to cross-react with human myosin and were visualized by a protein A-peroxidase method. Type I fibers were shown to contain slow myosin only, type IIA and IIB fibers fast myosin only, and type IIC fibers both myosins in various proportions. When muscle biopsies from well-trained athletes were investigated essentially the same staining pattern was observed. However, rarely occurring type I fibers with high glycolytic activity were detected containing additional small amounts of fast myosin and occasional type IIA fibers had small amounts of slow myosin. Based on the observation of various fiber types in which slow and fast myosin coexist we propose a dynamic continuum of fibers encompassing all fiber types.     

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 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.     

Analysis of myosin light and heavy chain types in single human skeletal muscle fibers

In this study, myosin types in human skeletal muscle fibers were investigated with electrophoretic techniques. Single fibers were dissected out of lyophilized surgical biopsies and typed by staining for myofibrillar ATPase after preincubation in acid or alkaline buffers. After 14C-labelling of the fiber proteins in vitro by reductive methylation, the myosin light chain pattern was analysed on two-dimensional gels and the myosin heavy chains were investigated by one-dimensional peptide mapping. Surprisingly, human type I fibers, which contained only the slow heavy chain, were found to contain variable amounts of fast myosin light chains in addition to the two slow light chains LC1s and LC2s. The majority of the type I fibers in normal human muscle showed the pattern LC1s, LC2s and LC1f. Further evidence for the existence in human muscle of a hybrid myosin composed of a slow heavy chain with fast and slow light chains comes from the analysis of purified human myosin in the native state by pyrophosphate gel electrophoresis. With this method, a single band corresponding to slow myosin was obtained; this slow myosin had the light chain composition LC1s, LC2s and LC1f. Type IIA and IIB fibers, on the other hand, revealed identical light chain patterns consisting of only the fast light chains LC1f, LC2f and LC3f but were found to have different myosin havy chains. On the basis of the results presented, we suggest that the histochemical ATPase normally used for fibre typing is determined by the myosin heavy chain type (and not by the light chains). Thus, in normal human muscle a number of 'hybrid' myosins were found to occur, namely two extreme forms of fast myosins which have the same light chains but different heavy chains (IIA and IIB) and a continuum of slow forms consisting of the same heavy chain and slow light chains with a variable fast light chain composition. This is consistent with the different physiological roles these fibers are thought to have in muscle contraction.     

去神经对快,慢肌纤维肌球蛋白ATPase影响的组织化学观察

本文用组织化学方法观察了豚鼠比目鱼肌(SOL)和腓骨第三肌(PT)在去神经后其快、慢纤维肌球蛋白ATPase特性的变化。在正常肌肉中Ⅰ型(慢)纤维和Ⅱ型(快)纤维分别具有酸和碱稳定ATPase活性。慢纤维在去神经后出现了碱稳定ATPase活性,而快纤维则无明显变化。结果表明,只有慢纤维的肌球蛋白ATPase特性才与神经支配有关。     

Fiber-type proportions in mammalian soleus muscle during postnatal development

We analyzed the fiber-type composition of the soleus muscle in rats and mice to determine whether the adult proportion of fiber types is fixed soon after birth or whether it changes during postnatal maturation. We examined muscles from animals varying in age from 1 week to 1 year using monoclonal antibodies that distinguish between fast and slow isoforms of myosin heavy chains. In cross sections of unfixed muscle containing profiles of all myofibers in the muscle, we counted the fibers that stained with antibodies to fast myosin, and in adjacent sections, those that stained positive with an antibody to slow myosin. We also counted the total number of fibers in each section. Rat soleus contained about 2500 myofibers, and mouse about 1000 at all ages studied, suggesting that myogenesis ceases in soleus by 1 week after birth or sooner. In mouse soleus, the relative proportions of fibers staining positive with fast and slow myosin antibodies were similar at all ages studied, about 60%–70% being fast and 30%–40% slow. In rat soleus, however, the proportions of fast antibody-positive and slow antibody-positive fibers changed dramatically during postnatal maturation. At 1 week after birth, about 50% of rat soleus fibers stained with fast myosin antibodies, whereas between 1 and 2 months this value fell to about 10%. In mouse, about 10% of fibers at 1 week, but none at 1 year, reacted with both fast and slow antibodies, whereas in rat, fewer than 3% bound both antibodies to a significant degree at 1 week. It is puzzling why, in rat soleus, the majority of apparently fast fibers present at 1 week is converted to a slow phenotype, whereas in mouse soleus the predominant change appears to be the suppression of fast myosin expression in a subset of fibers that expresses both myosin types at 1 week. It is possible that this may be related to differences in size and the amount of body growth between these two species.     

Fiber-type proportions in mammalian soleus muscle during postnatal development.

We analyzed the fiber-type composition of the soleus muscle in rats and mice to determine whether the adult proportion of fiber types is fixed soon after birth or whether it changes during postnatal maturation. We examined muscles from animals varying in age from 1 week to 1 year using monoclonal antibodies that distinguish between fast and slow isoforms of myosin heavy chains. In cross sections of unfixed muscle containing profiles of all myofibers in the muscle, we counted the fibers that stained with antibodies to fast myosin, and in adjacent sections, those that stained positive with an antibody to slow myosin. We also counted the total number of fibers in each section. Rat soleus contained about 2500 myofibers, and mouse about 1000 at all ages studied, suggesting that myogenesis ceases in soleus by 1 week after birth or sooner. In mouse soleus, the relative proportions of fibers staining positive with fast and slow myosin antibodies were similar at all ages studied, about 60%-70% being fast and 30%-40% slow. In rat soleus, however, the proportions of fast antibody-positive and slow antibody-positive fibers changed dramatically during postnatal maturation. At 1 week after birth, about 50% of rat soleus fibers stained with fast myosin antibodies, whereas between 1 and 2 months this value fell to about 10%. In mouse, about 10% of fibers at 1 week, but none at 1 year, reacted with both fast and slow antibodies, whereas in rat, fewer than 3% bound both antibodies to a significant degree at 1 week. It is puzzling why, in rat soleus, the majority of apparently fast fibers present at 1 week is converted to a slow phenotype, whereas in mouse soleus the predominant change appears to be the suppression of fast myosin expression in a subset of fibers that expresses both myosin types at 1 week. It is possible that this may be related to differences in size and the amount of body growth between these two species.     

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.     

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.     

Expression of myosin isoforms during notexin-induced regeneration of rat soleus muscles

Myosin isozymes and their fiber distribution were studied during regeneration of the soleus muscle of young adult (4-6 week old) rats. Muscle degeneration and regeneration were induced by a single subcutaneous injection of a snake toxin, notexin. If reinnervation of the regenerating muscle was allowed to occur (functional innervation nearly complete by 7 days), then fiber diameters continued to increase and by 28 days after toxin treatment they attained the same values as fibers in the contralateral soleus. If the muscles were denervated at the time of toxin injection, the early phases of regeneration still took place but the fibers failed to continue to increase in size. Electrophoresis of native myosin showed multiple bands between 3 and 21 days of regeneration which could be interpreted as indicating the presence of embryonic, neonatal, fast and slow myosins in the innervated muscles. Adult slow myosin became the exclusive from in innervated regenerates. In contrast, adult fast myosin became the predominant form in denervated regenerating muscles. Immunocytochemical localization of myosin isozymes demonstrated that in innervated muscles the slow form began to appear in a heterogeneous fashion at about 7 days, and became the major form in all fibers by 21-28 days. Thus, the regenerated muscle was almost entirely composed of slow fibers, in clear contrast to the contralateral muscle which was still substantially mixed. In denervated regenerating muscles, slow myosin was not detected biochemically or immunocytochemically whereas fast myosin was detected in all denervated fibers by 21-28 days. The regenerating soleus muscle therefore is clearly different from the developing soleus muscle in that the former is composed of a uniform fiber population with respect to myosin transitions. Moreover the satellite cells which account for the regeneration process in the soleus muscle do not appear to be predetermined with respect to myosin heavy chain expression, since the fibers they form can express either slow or fast isoforms. The induction of the slow myosin phenotype is entirely dependent on a positive, extrinsic influence of the nerve.     

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.     

Effects of hindlimb unweighting and aging on rat semimembranosus muscle and myosin.

We tested the hypothesis that lower specific force (force/cross-sectional area) generated by type II fibers from hindlimb-unweighted rats resulted from structural changes in myosin (i.e., a change in the ratio of myosin cross bridges in the weak- and strong-binding state during contraction). In addition, we determined whether those changes were age dependent. Permeabilized semimembranosus muscle fibers from young adult and aged rats, some of which were hindlimb unweighted for 3 wk, were studied for Ca(2+)-activated force generation and maximal unloaded shortening velocity. Fibers were also spin labeled specifically at myosin Cys707 to assess the structural distribution of myosin during maximal isometric contraction using electron paramagnetic resonance spectroscopy. Myosin heavy chain isoform (MHC) expression and the ratio of MHC to actin were evaluated in each fiber. Fibers from the unweighted rats generated 34% less specific force than fibers from weight-bearing rats (P < 0.001), independent of age. Electron paramagnetic resonance analyses showed that the fraction of myosin heads in the strong-binding structural state during contraction was 11% lower in fibers from the unweighted rats (P = 0.019), independent of age. More fibers from unweighted rats coexpressed MHC IIB-IIX compared with fibers from weight-bearing rats (P = 0.049). Unweighting induced a slowing of maximal unloaded shortening velocity and an increase in the ratio of MHC to actin in fibers from young rats only. These data indicate that altered myosin structural distribution during contraction and a preferential loss of actin contribute to unweighting-induced muscle weakness. Furthermore, the age of the rat has an influence on some parameters of changes in muscle contractility that are induced by unweighting.     

Coexistence of fast and slow types of myosin light chains in a single fiber of rat soleus muscle

Single muscle fibers were isolated from soleus and extensor digitorum longus muscle of adult rats. The muscle fiber type of single fibers was determined physiologically by the skinned fiber method according to the sensitivity to strontium (Sr) ions. The fiber type of single fibers was contrasted to the pattern of myosin light chains analyzed by one and two dimensional gel-electrophoreses. All the type 2 fibers isolated from soleus muscle contained both fast and slow types of myosin light chains.     

Evidence for expression of a common myosin heavy chain phenotype in future fast and slow skeletal muscle during initial stages of avian embryogenesis

We have utilized a key biochemical determinant of muscle fiber type, myosin isoform expression, to investigate the initial developmental program of future fast and slow skeletal muscle fibers. We examined myosin heavy chain (HC) phenotype from the onset of myogenesis in the limb bud muscle masses of the chick embryo through the differentiation of individual fast and slow muscle masses, as well as in newly formed myotubes generated in adult muscle by weight overload. Myosin HC isoform expression was analyzed by immunofluorescence localization with a battery of anti-myosin antibodies and by electrophoretic separation with SDS-PAGE. Results showed that the initial myosin phenotype in all skeletal muscle cells formed during the embryonic period (until at least 8 days in ovo) consisted of expression of a myosin HC which shares antigenic and electrophoretic migratory properties with ventricular myosin and a distinct myosin HC which shares antigenic and electrophoretic migratory properties with fast skeletal isomyosin. Similar results were observed in newly formed myotubes in adult muscle. Future fast and slow muscle fibers could only be discriminated from each other in developing limb bud muscles by the onset of expression of slow skeletal myosin HC at 6 days in ovo. Slow skeletal myosin HC was expressed only in myotubes which became slow fibers. These findings suggest that the initial commitment of skeletal muscle progenitor cells is to a common skeletal muscle lineage and that commitment to a fiber-specific lineage may not occur until after localization of myogenic cells in appropriate premuscle masses. Thus, the process of localization, or events which occur soon thereafter, may be involved in determining fiber type.     

ATP consumption and efficiency of human single muscle fibers with different myosin isoform composition

Chemomechanical transduction was studied in single fibers isolated from human skeletal muscle containing different myosin isoforms. Permeabilized fibers were activated by laser-pulse photolytic release of 1.5 mM ATP from p(3)-1-(2-nitrophenyl)ethylester of ATP. The ATP hydrolysis rate in the muscle fibers was determined with a fluorescently labeled phosphate-binding protein. The effects of varying load and shortening velocity during contraction were investigated. The myosin isoform composition was determined in each fiber by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At 12 degrees C large variations (three- to fourfold) were found between slow and fast (2A and 2A-2B) fibers in their maximum shortening velocity, peak power output, velocity at which peak power is produced, isometric ATPase activity, and tension cost. Isometric tension was similar in all fiber groups. The ATP consumption rate increased during shortening in proportion to shortening velocity. At 12 degrees C the maximum efficiency was similar (0.21-0.27) for all fiber types and was reached at a higher speed of shortening for the faster fibers. In all fibers, peak efficiency increased to approximately 0.4 when the temperature was raised from 12 degrees C to 20 degrees C. The results were simulated with a kinetic scheme describing the ATPase cycle, in which the rate constant controlling ADP release is sensitive to the load on the muscle. The main difference between slow and fast fibers was reproduced by increasing the rate constant for the hydrolysis step, which was rate limiting at low loads. Simulation of the effect of increasing temperature required an increase in the force per cross-bridge and an acceleration of the rate constants in the reaction pathway.     

Use of type-specific antimyosins to demonstrate the transformation of individual fibers in chronically stimulated rabbit fast muscles

Continuous stimulation of a rabbit fast muscle at 10 Hz changes its physiological and biochemical parameters to those of a slow muscle. These transformations include the replacement of myosin of one type by myosin of another type. Two hypotheses could explain the cellular basis of these changes. First, if fibers were permanently programmed to be fast or slow, but not both, a change from one muscle type to another would involve atrophy of one fiber type accompanied by de novo appearance of the other type. Alternatively, preexisting muscle fibers could be changing from the expression of one set of genes to the expression of another. Fluorescein-labeled antibodies against fast (AF) and slow (AS) muscle myosins of rabbits have been prepared by procedures originally applied to chicken muscle. In the unstimulated fast peroneus longus muscle, most fibers stained only with AF; a small percentage stained only with AS; and no fibers stained with both antibodies. In stimulated muscles, most fibers stained with both AF and AS; with increasing time of stimulation, there was a progressive decrease in staining intensity with AF and a progressive increase in staining intensity with AS within the same fibers. These results are consistent with a theory that individual preexisting muscle fibers can actually switch from the synthesis of fast myosin to the synthesis of slow myosin.