The ultrastructural location of C-protein,X-protein and H-protein in rabbit muscle

Summary Purified antibodies to the thick filament accessory proteins, C-protein, X-protein and H-protein, have been used to label fibres of three rabbit muscles, psoas (containing mainly fast white fibres), soleus (containing mainly slow red fibres) and plantaris (a muscle of mixed fibre type) and their location has been examined by electron microscopy.These accessory proteins are present on one or more of a set of eleven transverse stripes about 43 nm apart that have been observed previously in each half A-band. Each protein has a limited set of characteristic distributions. H-protein is present on stripe 3 (counting from the M-line) in the majority of psoas fibres but is absent in soleus and plantaris muscle. C-protein can occur (1) on stripes 4–11 (the commonest pattern seen in psoas); (2) on stripes 5–11 (in psoas and plantaris); (3) on stripe 3 together with stripes 5–11 (in plantaris); or (4) on none (in red fibres of all three muscles). X-protein can occur (1) on stripes 3–11 in the red fibres of all three muscles; (2) on stripe 4 only (in psoas and plantaris); (3) on stripes 3 and 4 (in psoas and plantaris) or (4) on none. Stripes labelled with anti-X are wider than those labelled with anti-C and consist of a doublet with an internal spacing of 16 nm. The patterns for the three accessory proteins, while overlapping, are in no case identical; this suggests the proteins do not simply substitute for one another.The precise axial positions of the anti-C labelled stripes differ from those of the anti-X stripes; the anti-X stripes lie about 8–9 nm further from the M-line than the corresponding anti-C stripes. This implies that the inner member of an X-protein doublet lies in a very similar position to a C-protein stripe. The anti-H labelled stripe seen in most psoas fibres lies 14 nm nearer the M-line than stripe 3 of the anti-X labelled array in psoas red fibres and is staggered from a continuation of the C-protein array by about 4 nm.The labelling patterns were constant within a fibre and suggest a very precise assembly mechanism. The number of classes of fibre, as defined by the accessory proteins present and their arrangement, exceeds the number of fibre types presently recognized.     

Compared properties of the contractile system of skinned slow and fast rat muscle fibres

Chemically skinned fibres from soleus and plantaris rat muscles were used to compare the contractile properties of slow and fast muscles. The maximal isometric tension appeared larger in plantaris than in soleus fibres. The apparent Ca²⁺ threshold for activation was lower in slow than in fast fibres while Ca²⁺ concentrations required to obtain either the maximal tension or half maximal tension (pCa₅₀) were lower in fast than in slow fibres. This apparent difference in Ca²⁺ sensitivity will be discussed. As could be expected from other studies, a faster force development in plantaris than in soleus fibres occurred. However, one interesting new result showed that in soleus, the kinetics of the tension development estimated by the t _max parameter were slightly dependent on the Ca²⁺ concentration whereas the t ₅₀ parameter changed significantly with the Ca²⁺ concentration. In plantaris, both t _max and t ₅₀ parameters were found to depend strongly on the Ca²⁺ concentration. Finally, the plantaris muscle showed a greater caffeine sensitivity than the soleus muscle. All the results suggested that the Ca-regulatory mechanism in the slow fibres was essentially different from that in the fast fibres.     

Cytoplasm-to-myonucleus ratios in plantaris and soleus muscle fibres following hindlimb suspension

Summary In multinucleated skeletal muscle fibres the size of the cytoplasmic volume-to-myonucleus ratio is related to the myosin heavy chain phenotype, with the ratio being larger in those fibres expressing the fast myosin heavy chain phenotype. It is unknown, however, whether this ratio is modulated during muscle fibre adaptation, such as that which occurs following muscle unloading. In this study the relationship between cross sectional area, myonuclear number and myosin type, in single fibres from the plantaris and soleus muscles of adult rats following 28 days of hindlimb suspension was examined. Each fibre was cut transversely into two segments; one segment was used for immunohistochemical identification of myosin type, the other for determination of cross sectional area and myonuclei number. Single fibre analysis revealed significant atrophy of both plantaris fast and soleus slow fibres; the mean cross sectional area (m²) of these fibres, 3104±183 and 2082±107 (mean ±se), being 70 and 45%, respectively, of control means. The decreases in cross sectional area were not accompained by corresponding decreases in the number of myonuclei (myonuclei/mm); in plantaris fast fibres the mean myonuclei counts were within the control range (88±8 (hindlimb suspension), 76±7 (control)), in soleus slow fibres the counts were significantly increased (185±12 (hindlimb suspension), 154±11 (control)). The changes resulted in a significant decrease in the cytoplasmic volume-to-myonucleus ratio (m³×10³) for both fibre types; the mean ratios of 39±3 and 12±1, were 60% and 36% of control means for the plantaris fast and soleus slow fibres, respectively. These results indicate that following hindlimb suspension atrophy of muscle fibres the myonuclei numbers remain constant or increase and, hence, the effective cytoplasmic-to-myonucleus ratio is decreased. Further, the described changes are significantly greater in soleus slow than plantaris fast fibres.     

Subcellular localization of newly incorporated myosin in rabbit fast skeletal muscle undergoing stimulation-induced type transformation

Summary Immunogold labelling was used to study the distribution of newly synthesized slow muscle myosin (SM) at the ultrastructural level as it replaced fast muscle myosin (FM) in rabbit muscles undergoing stimulation-induced type transformation. Control fast muscle was labelled strongly with antibody to FM and control slow muscle with antibody to SM; label was confined to the A-band. Well-defined differences in the distribution of label within the A-band suggested that the monoclonal antibodies used corresponded to epitopes on different parts of the myosin molecule; this was confirmed by Western blots of subfragments prepared from FM and SM. After 4 weeks of continuous stimulation at 10 Hz, fibres of the tibialis anterior muscle reacted with antibodies to both isoforms; after 6 weeks, labelling was obtained only with antibody to SM. After a 7-week period of stimulation and 3 further weeks of recovery, fibres again reacted with both antibodies. In all positively-labelled sections, the distribution of gold particles was characteristic of the antibody and independent of the origin or history of the fibres. This observation supports the conclusion that newly synthesized myosin is capable of being incorporated throughout the length and cross-section of the A-band.     

Avian adductor profundus muscle: characterization of a pure slow tonic region by histochemical,monoclonal antibody and peptide mapping studies

Summary The fibre type composition of the avian adductor profundus (AP) muscle which is composed of a thick white posterior part (Post. AP) and a thin red anterior part (Ant. AP) was investigated. Using the histochemical ATPase technique, monoclonal antibody analysis of myosin and C-protein isoforms, and electrophoretic and peptide mapping analyses of myosin, we have established that the Post. AP is composed of essentially pure slow tonic fibres similar to those of the anterior latissimus dorsi muscle (ALD). The Ant. AP, on the other hand, is shown to contain a mixture of slow and fast fibres, the latter giving immunocytochemical reactions atypical of the fast fibres. The larger size of the Post. AP in comparison with the ALD muscle should provide significantly more tissue for biochemical studies of tonic fibres than was previously available.     

Expression of slow and fast myosin heavy chains in overloaded muscles of the developing rat

Summary The present study examines the developmental accumulation of slow myosin heavy chain in the extensor digitorum longus, soleus and plantaris muscles of rats after early post-natal imposition of mechanical overload by removal of synergistic muscles. The proportions of slow and fast myosin heavy chain were measured in each muscle by ELISA. Fibres expressing slow myosin were examined immunocytochemically using a monoclonal antibody specific for slow MHC. Between 30 and 60 days of age, MHC increases by 15% (p<0.001) in the soleus and by 27% (p<0.001) in the plantaris of normally developing, unoperated animals. The effect of overload on the soleus and plantaris is to accelerate the rate of increase in slow MHC accumulation so that levels are respectively 16 and 39% higher than controls by 30 days of age (p<0.001). By 60 days, the control soleus and plantaris attain levels of slow MHC roughly equivalent to their overloaded counterparts. In overloaded plantaris, the increase in levels of slow myosin does not occur at the expense of fast myosin expression. In the EDL there is a normal developmentally regulated decrease in slow MHC accumulation, reflected by a 40% decrease in levels of slow MHC (p<0.0001) and a 50% decrease in the number of slow fibres (p<0.001), between 30 days and 20 weeks of age. This elimination of slow myosin accumulation in the EDL is unimpeded by chronic overload. Thus, muscles react to mechanical overload in a tissue specific manner. The pattern of response is conservative and potentiates normal, long term maturational shifts in myosin heavy chain expression characteristic of each muscle.     

Characterization of the cross-bridge force-generating step using inorganic phosphate and BDM in myofibrils from rabbit skeletal muscles

The inhibitory effects of inorganic phosphate (P_i) on isometric force in striated muscle suggest that in the ATPase reaction P_i release is coupled to force generation. Whether P_i release and the power stroke are synchronous events or force is generated by an isomerization of the quaternary complex of actomyosin and ATPase products (AM.ADP.P_i) prior to the following release of P_i is still controversial. Examination of the dependence of isometric force on [P_i] in rabbit fast (psoas; 5-15 °C) and slow (soleus; 15-20 °C) myofibrils was used to test the two-step hypothesis of force generation and P_i release. Hyperbolic fits of force-[P_i] relations obtained in fast and slow myofibrils at 15 °C produced an apparent asymptote as [P_i]∞ of 0.07 and 0.44 maximal isometric force (i.e. force in the absence of P_i) in psoas and soleus myofibrils, respectively, with an apparent K _d of 4.3 m m in both. In each muscle type, the force-[P_i] relation was independent of temperature. However, 2,3-butanedione 2-monoxime (BDM) decreased the apparent asymptote of force in both muscle types, as expected from its inhibition of the force-generating isomerization. These data lend strong support to models of cross-bridge action in which force is produced by an isomerization of the AM.ADP.P_i complex immediately preceding the P_i release step.     

Correlation of dystrophin–glycoprotein complex and focal adhesion complex with myosin heavy chain isoforms in rat skeletal muscle

Aim: The dystrophin–glycoprotein complex (DGC) and focal adhesion complex (FAC) are transmembrane structures in muscle fibres that link the intracellular cytoskeleton to the extracellular matrix. DGC and FAC proteins are abundant in slow‐type muscles, indicating the structural reinforcement which play a pivotal role in continuous force output to maintain posture for long periods. The aim of the present study was to examine the expression of these structures across fast‐type muscles containing different myosin heavy chain (MHC) isoform patterns which reflect the fatigue‐resistant characteristics of skeletal muscle. Methods: We measured the expression of dystrophin and β1 integrin (representative proteins of DGC and FAC respectively) in plantaris, extensor digitorum longus, tibialis anterior, red and white portions of gastrocnemius, superficial portion of vastus lateralis and diaphragm, in comparison with soleus (SOL) and cardiac muscle from rats. Results: The expression of dystrophin and β1 integrin correlated positively with the percentage of type I, IIa and IIx MHC isoforms and negatively with that of type IIb MHC isoform in fast‐type skeletal muscles, and their expression was abundant in SOL and cardiac muscle. Conclusion: Our results support the idea that DGC and FAC are among the factors that explain the fatigue‐resistant property not only of slow‐type but also of fast‐type skeletal muscles.     

Maximum shortening velocity and coexistence of myosin heavy chain isoforms in single skinned fast fibres of rat skeletal muscle

Summary Myosin heavy chain composition of a large number (288) of single fibres from slow (soleus), and fast (superficial part of tibialis anterior, and plantaris) muscles of adult (3–5-month-old) Wistar rats was determined. A combination of SDS-PAGE and monoclonal antibodies against myosin heavy chains allowed to identify four myosin heavy chain isoforms (1, 2A, 2X, and 2B) and to detect myosin heavy chain coexistence. Four groups of fibres containing only one myosin heavy chain (1 myosin heavy chain, 2A myosin heavy chain, 2X myosin heavy chain, and 2B myosin heavy chain), and five groups containing more than one myosin heavy chain 1 and 2A myosin heavy chains, 2A and 2X myosin heavy chains, 2X and minor amounts of 2B (2X-2B fibres), 2B and minor amounts of 2X (2B-2X fibres), and 2A, 2X, and 2B myosin heavy chain were identified and their relative percentages were assessed. Coexistence of fast myosin heavy chain isoforms was found to be very frequent (50% of the fibres in plantaris, and 30% in tibialis anterior), whereas coexistence of slow and fast (2A) myosin heavy chain was very rare. Maximum shortening velocity (V₀) was determined using the slack-test procedure in a subset of 109 fast fibres from the above population. The values of V₀ formed a continuum extending from 2A to 2X to 2X-2B to 2B-2X to 2B fibres. 2A fibres had the lowest value of V₀ and 2B fibres the highest. Only the differences between 2A and 2B and 2A and 2B-2X fibres were statistically significant. Importantly, the variability of V₀ in fibres containing only one myosin heavy chain and in fibres containing a variable proportion of two myosin heavy chain isoforms was similar and, in some case (e.g. 2B fibres), such to encompass the whole range of variation of fast fibres shortening velocities. The results of this study demonstrate that the large variability in maximum shortening velocity of fast fibres is not due to myosin heavy chain coexistence, and therefore suggest that it cannot be explained on the basis of myosin heavy chain composition.     

The effect of calcium on the maximum velocity of shortening in skinned skeletal muscle fibres of the rabbit

Summary The relationship between maximum shortening velocity (V _max) and free calcium concentration has been studied in skinned single fibres from rabbit psoas and soleus muscles. At both 10 and 15° C,V _max measured in the psoas fibres was found to decrease by 40% when the pCa (-log[Ca²⁺]) was increased from 5.49 (maximally activating) to 6.21. Further decreases inV _max were observed when the pCa was increased to 6.32.V _max measured in soleus fibres at 15° C also decreased when the Ca²⁺ concentration was lowered, though the magnitude of this effect was slightly less than in the psoas fibres. Thus, a distinct effect of Ca²⁺ uponV _max has been shown to occur in mammalian skeletal muscle. The occurrence of this effect in both fast and slow muscle types may indicate that the underlying mechanism in the two cases is similar.     

Immunocytochemical and electrophoretic analyses of changes in myosin gene expression in cat limb fast and slow muscles during postnatal development

Summary Changes in myosin synthesis during the postnatal development of the fast extensor digitorum longus (EDL) and the slow soleus muscles of the kitten were examined using immunocytochemical techniques supplemented by pyrophosphate gel electrophoresis and gel electrophoresis-derived enzyme linked immunosorbent assay (GEDELISA) of myosin isoforms. The antibodies used were monoclonals against heavy chains of slow and fast myosins and a polyclonal against foetal/embryonic myosin. In both muscles in the newborn kitten, there was a population of more mature fibres which stained strongly for slow but weakly for foetal/embryonic myosin. These fibres were considered to be primary fibres. They formed 4.8% of EDL fibres and 26% of soleus fibres at birth, and continued to express slow myosin in adult muscles. The less mature secondary fibres stained strongly for foetal/embryonic myosin, and these could be divided into two subpopulations; fast secondaries in which foetal/embryonic myosin was replaced by fast myosin, and slow secondaries in which the myosin was replaced by slow myosin. At 50 days the EDL had a large population of fast secondaries (83% of total fibres) and a small population of slow secondaries which gradually transformed into fast fibres with maturity. The vast majority of secondary fibres in the soleus were slow secondaries, in which slow myosin synthesis persisted in adult life. There was a restricted zone of fast secondaries in the soleus, and these gradually transformed into slow fibres in adult life. It is proposed that the emergence of primary fibres and the two populations of secondary fibres is myogenically determined.     

Are region-specific changes in fibre types attributable to nonuniform muscle hypertrophy by overloading?

Muscle fibre composition was compared among the proximal (25%), middle (50%) and distal (75%) regions of the muscle length to investigate whether compensatory overload by removal of synergists induces region-specific changes of fibre types in rat soleus and plantaris muscles. In addition, we evaluated fibre cross-sectional area in each region to examine whether fibre recruitment pattern against functional overload is nonuniform in different regions. Increases in muscle mass and fibre area confirmed a significant hypertrophic response in the overloaded soleus and plantaris muscles. Overloading increased the percentage of type I fibres in both muscles and that of type IIA fibres in the plantaris muscle, with the greater changes being found in the middle and distal regions. The percentage of type I fibres in the proximal region was higher than that of the other regions in the control soleus muscle. In the control plantaris muscle, the percentage of type I and IIA fibres in the middle region were higher than that of the proximal and distal regions. With regard to fibre size, type IIB fibre area of the middle and distal regions in the plantaris increased by 51% and 57%, respectively, with the greater changes than that of the proximal region (37%) after overloading. These findings suggest that compensatory overload promoted transformation of type II fibres into type I fibres in rat soleus and plantaris muscles, with the greater changes being found in the middle and distal regions of the plantaris muscle.     

Hyperbaric exposure with high oxygen concentration enhances oxidative capacity of neuromuscular units

The effects of hyperbaric exposure with high oxygen concentration on spinal motoneurons and the skeletal muscle fibers that they innervate were investigated. Five-week-old male rats were exposed to a hyperbaric (1.25 atmospheric pressure) environment with a high oxygen concentration (35.0%) for 6h daily. The number, cell body size, and oxidative enzyme activity of motoneurons innervating the soleus and plantaris muscles were examined after 8 weeks of hyperbaric exposure. In addition, the fiber type distribution, cell size, and oxidative enzyme activity of the slow soleus and fast plantaris muscles were examined. The oxidative enzyme activity of alpha motoneurons innervating the soleus and plantaris muscles increased after hyperbaric exposure, irrespective of their cell body sizes. The percentage of high-oxidative fibers in the soleus and plantaris muscles increased after hyperbaric exposure. The oxidative enzyme activity of all types of fibers in the soleus and plantaris muscles increased after hyperbaric exposure. It is concluded that hyperbaric exposure with high oxygen concentration enhances the oxidative capacity of neuromuscular units.     

Distribution of isoforms of the myofibrillar proteins in myoid cells of thymus

Summary Myoid cells of calf and rat thymus have been identified by staining with a monoclonal antibody to the heavy chain of myosin that is not isoform specific. Heterogeneity in the protein composition of myoid cells has been demonstrated by staining with antibodies to the skeletal muscle isoforms of the myosin heavy chain, C-protein and components of the troponin complex. The immunochemical studies suggest that the myoid cells contain proteins closely resembling if not identical with those present in the myofibrils of skeletal muscle. The slow and fast skeletal muscle isoforms of the myofibrillar proteins are present in a large proportion of the myoid cells. A fraction of the myoid cells contains only the fast isoforms of the myofibrillar proteins but there is no sharp compartmentalization of the isoforms as occurs in type 1 and type 2 fibres of skeletal muscle. In general the pattern of gene expression is similar to that of developing skeletal muscle.     

Differential modification of myosin heavy chain expression by tenotomy in regenerating fast and slow muscles of the rat

We have examined the effect of tenotomy on the expression of myosin heavy chains (MyHC) in regenerating fast and slow skeletal muscles. Degeneration/regeneration of the left soleus and plantaris of Wistar male rats was induced by an injection into the muscle belly of a myotoxin (snake venom: Notechis scutatus scutatus). MyHC isoform content of regenerating plantaris and soleus muscles were studied 21 days after muscle injury using an electrophoretic technique. Tenotomy of the regenerating plantaris (mechanical underload) did not alter its MyHC expression (P > 0.05). In contrast, tenotomy of the regenerating soleus increased its relative levels of MyHC-2b (P < 0.05) and MyHC-2x/d (P < 0.01), and decreased its relative level of MyHC-1 (P < 0.01). Tenotomy of the synergistic gastrocnemius (overload) tended to decrease the relative level of MyHC-2b in regenerating plantaris (P < 0.07). The effect of tenotomy of the synergistic gastronecmius on the regenerating soleus was different: a decrease in the relative levels of MyHC-1 (P < 0.05) and an increase in the relative level of MyHC-neonatal (P < 0.01). In conclusion, and in contrast to a regenerating slow muscle, a change of mechanical loading by tenotomy did not seem to markedly alter the expression of mature MyHC phenotype in a fast regenerating muscle.     

Accumulation of caesium and rubidium in vivo by red and white muscles of the rat

1. Rats were given drinking water containing either 20 mM-CsCl or 20 mM-RbCl for a period of 2 weeks. Samples of blood were then taken from the rats under anaesthetic. They were immediately centrifuged and the plasma taken for analysis. Soleus muscles, diaphragm, extensor digitorum longus, white gastrocnemius and vastus lateralis muscles were then taken from the dead animals and these and the plasma were analysed for potassium, and for caesium or rubidium by means of the flame photometer.2. The concentrations of potassium and rubidium or caesium in the fibre water of these various muscles and in the samples of plasma water were then calculated.3. It was found that the red muscles including soleus and diaphragm generally tended to accumulate caesium and rubidium to a greater extent than did the white muscles such as the white gastrocnemius and vastus lateralis.4. When the concentration ratio [K](i)/[K](o) was divided into the ratio [Rb](i)/[Rb](o) for the different muscles, values of about 1.3 were obtained for the red muscles compared with values about 1.14 for white muscles.5. When in the case of the caesium-treated rats the ratio [K](i)/[K](o) was divided into the ratio [Cs](i)/[Cs](o) values ranged from 1.94 +/- 0.12 for the red soleus to 1.08 +/- 0.09 for the white gastrocnemius.6. When these values in the caesium-treated animals were plotted against the percentage of red fibres in the five muscle types (as obtained from the data of Sreter & Woo, 1963) the graph indicated that the white fibres had similar ionic gradients for Cs(+) and K(+) and that affinity for Cs(+) was confined to the red fibres.7. The membrane potential measured in soleus and extensor muscles immersed in plasma from the same animal was not significantly different from E(K) but was much less than E(Cs).8. These results are interpreted in terms of permeability differences between the slow red fibres and white twitch fibres.     

Ageing influences myonuclear domain size differently in fast and slow skeletal muscle of rats

Aim: In multinucleated skeletal muscle, a myonuclear domain is the region of cytoplasm governed by one nucleus, and myofibres are mosaics of overlapping myonuclear domains. Association of ageing and myonuclear domain is important in the understanding of sarcopenia and with prevention or combating age‐related muscle declines. This study examined the effects of age, fibre type and muscle on nucleo‐cytoplasmic (N/C) relationships as reflecting myonuclear domain size. Methods: The N/C was compared in fibre types of soleus and plantaris muscles from young (n = 6) and ageing (n = 8) male Fisher 344 rats. Results: There were no significant differences in fibre type composition or cross‐sectional area of the soleus across ages. The old soleus had significantly more myonuclei, resulting in a significantly smaller myonuclear domain size. The plantaris muscle showed a higher percentage of slow fibres in old compared with young fibres. There were no differences in the number of myonuclei or in myonuclear domain size between young and older animals. Conclusion: We found muscle‐specific differences in the effects of ageing on myonuclear domain, possibly as a result of reduced efficiency of the myonuclei in the slow muscles.     

The role of neural and mechanical influences in maintaining normal fast and slow muscle properties

The relative importance of neural and mechanical influences in maintaining normal slow and fast muscle properties remains unclear. To address this issue, we studied the effects of 10 days of hindlimb unloading (HU) with or without tenotomy and/or denervation on the cross-sectional area (CSA), myosin heavy chain (MHC) expression (immunohistochemistry) and composition (gel electrophoresis), and myonuclear number in soleus and plantaris fibers in adult male Wistar rats. In general, the adaptations in fiber type and size were similar using either single fiber gel or immunohistochemical analyses. HU resulted in atrophy of type I and I+IIa/x MHC fibers in the soleus and in type I, I+IIa/x, IIa/x, IIa/x+IIb, and IIb MHC fibers in the plantaris. Addition of tenotomy and/or denervation in HU rats had minimal effects on fiber CSA in the soleus, but fiber CSA in the plantaris further decreased, particularly in fibers expressing only fast MHCs. HU resulted in a de novo appearance of type I+IIa/x+IIb and IIa/x+IIb MHC fibers in the soleus and of type I+IIa/x+IIb MHC fibers in the plantaris.Tenotomy and/or denervation in HU rats had no further effect on the fiber type composition of either muscle. Mean myonuclear number/mm of type I fibers was decreased in the soleus of HU rats, and increased in type I and I+IIa/x fibers in HU plus tenotomy (HU+Ten) rats. In the plantaris, mean myonuclear number/mm of type IIa/x, IIa/x+IIb, and IIb fibers was lower after HU with or without tenotomy and/or denervation. Mean cytoplasmic volume/myonucleus ratio of type I and I+IIa/x fibers in the soleus of the HU group tended to be smaller than in controls. The largest decrease was noted in the HU+Ten group. In the plantaris, this ratio was unaffected by HU alone, but was decreased by addition of tenotomy and/or denervation when all fiber types were combined. These data indicate that the major cause of fiber atrophy and adaptations in myonuclear domain size in the slow soleus of HU rats is the chronic reduction in force generation, whereas the elimination of neuromuscular contact via denervation results in additional fiber atrophy and adaptations in myonuclear domain size in the fast plantaris.