Expression of a fast myosin heavy chain mRNA in individual rabbit skeletal muscle fibers with intermediate oxidative capacity.

In situ hybridization (ISH) of myosin heavy chain (MHC) mRNA, immunofluorescent detection of MHC protein, and oxidative enzyme histochemistry were performed on the same fibers in serially sectioned rabbit skeletal muscle. By combining these three techniques quantitatively, on a fiber-by-fiber basis, fibers that expressed mRNA complementary to a fast MHC cDNA pMHC24-79 of unknown subtype (Maeda et al., 1987) were classified into fiber types with respect to slow myosin expression and oxidative capacity. As expected, slow fibers had low hybridization to pMHC24-79. Fast fibers were divided into three subtypes. mRNA from the low oxidative fibers (fast-glycolytic, IIB) did not hybridize with pMHC24-79. Fast fibers whose mRNA hybridized best to pMHC24-79 were mainly in the intermediate range of oxidative capacity (probably IIX). The fast fibers with the highest oxidative capacity had low hybridization to this MHC mRNA (probably IIA). Thus, pMHC24-79 was identified as a clone of a fast isomyosin, tentatively designated as the fast IIX with intermediate oxidative capacity. The expression of more than a single species of fast and slow isomyosin mRNAs in classically defined fiber type was considered in interpreting these results.     

Three myosin heavy chain isoforms in type 2 skeletal muscle fibres

Summary Mammalian skeletal muscles consist of three main fibre types, type 1, 2A and 2B fibres, with different myosin heavy chain (MHC) composition. We have now identified another fibre type, called type 2X fibre, characterized by a specific MHC isoform. Type 2X fibres, which are widely distributed in rat skeletal muscles, can be distinguished from 2A and 2B fibres by histochemical ATPase activity and by their unique staining pattern with seven anti-MHC monoclonal antibodies. The existence of the 2X-MHC isoform was confirmed by immunoblotting analysis using muscles containing 2X fibres as a major component, such as the normal and hyperthyroid diaphragm, and the soleus muscle after high frequency chronic stimulation. 2X-MHC contains one determinant common to 2B-MHC and another common to all type 2-MHCs, but lacks epitopes specific for 2A- and 2B-MHCs, as well as an epitope present on all other MHCs. By SDS-polyacrylamide gel electrophoresis 2X-MHC shows a lower mobility compared to 2B-MHC and appears to comigrate with 2A-MHC. Muscles containing predominantly 2X-MHC display a velocity of shortening intermediate between that of slow muscles and that of fast muscles composed predominantly of 2B fibres.     

The extent of amino-terminal heterogeneity in rabbit fast skeletal muscle troponin T

Summary The extent and nature of fast troponin T (TnT) heterogeneity has been assessed in rabbit skeletal muscle. Previous studies identified two major fast TnT species (TnT_1f and TnT_2f), in the fast white muscle erector spinae, differing in theirN-terminal cyanogen bromide (CNBr) fragments. Here a monoclonal antibody that recognizes a conserved region of TnT was used to characterize two additional TnT species (TnT_3f and TnT_4f) in the epaxial and limb musculature and a minor species (TnT_cf) in craniofacial muscles. A combination of CNBr peptide mapping, immunoblotting and specific labelling of theN-terminus shows that these TnT species also differ in theirN-terminal region. This observation is consistent with cDNA studies that predicted theN-terminal region is hypervariable. One additional species, a variant of TnT_2f present in the tongue, was identified by two-dimensional gel electrophoresis. The limited number of TnT variants indicates that the full potential for heterogeneity inferred from the cDNA studies is not realized. This conclusion is supported by immunoblot analysis with a monoclonal antibody that recognizes an epitope in the hypervariableN-terminal region which is present in all variants of TnT_1f and TnT_2f but absent from the lower molecular weight species TnT_3f and TnT_4f.     

Chromosomal distribution of genes coding for fast twitch skeletal muscle myosin light chains

The mouse fast twitch skeletal muscle myosin light chains are encoded by a multigene family which comprises the gene coding for the myosin light chain 2 (Myl_2f), and the gene coding for both myosin light chains 1 and 3 (Myl_1f/Myl_3f). In addition, a Myl_1f/Myl_3f-related pseudogene is present in the domestic mouse Mus musculus. The members of this gene family were assigned to chromosomes by molecular hybridization; using DNA extracted from a panel of cloned mouse-Chinese hamster somatic hybrid cells and specific DNA probes. The genes coding for the light chains of the myosin molecule are dispersed on several chromosomes, while genes coding for the heavy chain of myosin are located on a single, different chromosome.     

Dependence of cross-bridge kinetics on myosin light chain isoforms in rabbit and rat skeletal muscle fibres

Cross-bridge kinetics underlying stretch-induced force transients was studied in fibres with different myosin light chain (MLC) isoforms from skeletal muscles of rabbit and rat. The force transients were induced by stepwise stretches (< 0.3% of fibre length) applied on maximally Ca²⁺-activated skinned fibres. Fast fibre types IIB, IID (or IIX) and IIA and the slow fibre type I containing the myosin heavy chain isoforms MHC-IIb, MHC-IId (or MHC-IIx), MHC-IIa and MHC-I, respectively, were investigated. The MLC isoform content varied within fibre types. Fast fibre types contained the fast regulatory MLC isoform MLC2f and different proportions of the fast alkali MLC isoforms MLC1f and MLC3f. Type I fibres contained the slow regulatory MLC isoform MLC2s and the slow alkali MLC isoform MLC1s. Slow MLC isoforms were also present in several type IIA fibres. The kinetics of force transients differed by a factor of about 30 between fibre types (order from fastest to slowest kinetics: IIB > IID > IIA ≫ I). The kinetics of the force transients was not dependent on the relative content of MLC1f and MLC3f. Type IIA fibres containing fast and slow MLC isoforms were about 1.2 times slower than type IIA fibres containing only fast MLC isoforms. We conclude that while the cross-bridge kinetics is mainly determined by the MHC isoforms present, it is affected by fast and slow MLC isoforms but not by the relative content of MLC1f and MLC3f. Thus, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the cross-bridge kinetics.     

An age-related type IIB to IIX myosin heavy chain switching in rat skeletal muscle

A novel fast-twitch motor unit type, called the IIX-myosin heavy chain (MHC) motor unit, identified by the glycogen depletion technique together with a series of monoclonal antibodies (mAbs) specific for MHCs, has been isolated recently in the rat tibialis anterior muscle. In young animals, this unit has physiological, biochemical and morphometrical properties which separate it from the IIA- and IIB-MHC motor units. In old age, on the other hand, the IIX-MHC units display physiological, biochemical and morphometrical properties resembling the IIB-MHC motor units. Based on these results it was proposed that a transition from IIB to IIX motor units occurs during ageing. In an attempt to clarify this point, the MHC composition was identified by 6% SDS-PAGE and immunoblotting analysis, using specific mAbs antibodies, in the same fast-twitch tibialis anterior muscles in young (3–6 months, n = 9) and old (20–24 months, n = 16) rats from which the single motor units had been identified previously. The IIX-MHC comigrates together with the HA-MHC band in 6% SDS-PAGE and only two MHC bands are observed in the rat tibialis anterior muscle, i.e. the IIA-IIX-and IIB-MHC bands. A significant increase (P < 0.001) in the average relative amount of the HA-IIX —MHC was observed in the old (45 ± 17%) as compared to the young (23 ± 4%) animals, accompanied by a corresponding decrease in IIB-MHC content. It was demonstrated in immunoblotting analysis that only trace amounts of IIA-MHC were detectable in the IIA-IIX-MHC band in both young and old TA muscles, indicating a substantial increase in the IIX-MHC content in old age. Thus, the present results together with previous observations at the motor unit level strongly support an age-related motor unit transition from type IIB- to IIX-MHC.     

The reconstruction of myosin filaments in rabbit psoas muscle from solubilized myosin

Summary Using rabbit psoas muscle strips, A-bands with their myosin-containing thick filaments have been substantially reconstructedin situ (as judged by electron and light microscopy and by low-angle X-ray diffraction analysis) after prior solubilization of the myosin filaments in high ionic strength potassium phosphate solution. The maintenance of a very high local concentration of soluble myosin, by means of a closely apposed artificial semi-permeable membrane is necessary for reconstruction of full-length filaments. This reconstruction effect can be totally abolished by pre-glycerolation of the muscle, or (reversibly) by pre-depletion of Ca²⁺. Reconstruction at longer sarcomere lengths (>2.6m) is anomalous, part-length stub filaments being formed, with their stub tails projecting out from the I-Z-I lattice. A model is proposed to explain this reconstruction effect.     

Quasi-elastic light scattering studies of rabbit skeletal myosin solutions

Summary Homodyne measurements of the laser light spectrum scattered from solutions of rabbit skeletal muscle myosin in high ionic-strength media manifested a characteristicD value dependence on myosin concentrations. Using the typicalD versus myosin concentration curves obtained in the presence of 0.5m phosphate and 0.2m phosphate respectively as references, it has been shown that: (1) the observed phenomena are completely reversible; (2) minor components such as C- and F-protein do not significantly influence the measuredD values; and (3) the effect of preparation procedures on these dynamic light-scattering measurements is negligible. A common argument (irreversible aggregation) against a monomer-dimer equilibrium is ruled out; on the other hand, some doubt still remains with regard to the existence and physiological significance of a reversible dimerization.     

Correlation between percentage fiber type area and myosin heavy chain content in human skeletal muscle

Histochemical methods are routinely used to delineate skeletal muscle fiber types. In the present investigation, this qualitative determination of fiber type composition was compared to the electrophoretically determined myosin heavy chain (MHC) content from a large number of human muscle biopsy samples. Biopsies were taken from the vastus lateralis muscle at the beginning and every 2 weeks during 8 weeks of highi-ntensity resistance training from men (n = 13) and woman (n = 8). Muscle was also extracted from nontraining men (n = 7) and women (n = 5) at the same periods. Six muscle fiber types (I, IC, IIAC, IIA, IIAB, and IIB) were determined using basic myofibrillar adenosine triphosphatase histochemistry. Cross-sectional areas were determined for the three major fiber types (I, IIA, and IIB) and used to calculate the percentage area of these types. Electrophoretic techniques were used to separate and quantify the percentage MHC content in these same biopsy samples, and these data were then used to compare with the percentage fiber type area. Correlation analyses suggest a relationship between the histochemically assessed percentage fiber type area and the electrophoretically assessed MHC content in human limb musculature. However, because of possible histochemical misclassification of some fibers (especially in trained muscle) both techniques may be essential in yielding important information about fiber type composition and possible fiber type transformations.     

The effect of skeletal myosin light chain kinase gene ablation on the fatigability of mouse fast muscle

Contraction-induced activation of a skeletal muscle specific Ca(2+) and calmodulin dependent myosin light chain kinase (skMLCK) catalyzes phosphorylation of the myosin regulatory light chain (RLC), a reaction that potentiates twitch force. The purpose of this study was to test the effect of skMLCK gene ablation on the fatigability of mouse extensor digitorum longus (EDL) muscle (in vitro at 25°C). Muscles were isolated from wildtype (WT, n = 10-12) and skeletal MLCK knockout (skMLCK KO, n = 10-12) mice and fatigued using a protocol consisting of 5 min of repeated tetanic stimulation (150 Hz for 1000 ms every 5 s). Both twitch (P(t)) and tetanic (P(o)) force as well as unloaded shortening velocity (V(o)) were assessed before, during and after fatiguing stimulation. Fatiguing stimulation increased RLC phosphorylation in WT but not skMLCK KO muscles (16 ± 0.01-0.63 ± 0.02 and 0.07 ± 0.02-0.08 ± 0.02 mol phos mol RLC, respectively). Although P(t) was potentiated above baseline in both WT and KO muscles, this increase was greater in WT than in KO muscles (to 1.37 ± 0.05 vs. 1.14 ± 0.02 of unpotentiated values, respectively). The difference in P(t) persisted until P(o) had been diminished to ~60% of baseline and thereafter P(t) declined to similar levels in both WT and KO muscles (to ~35% of initial). Overall, the time-course and decline in P(o) for WT and KO was similar (reduced to 0.20 ± 0.01 and 0.20 ± 0.01 of baseline, respectively) (P < 0.05). Initial values for V(o) were similar between WT and KO muscles and, moreover, the fatigue related decline in Vo was similar for both muscle genotypes (P < 0.05). Thus, our results demonstrate that skMLCK--catalyzed RLC phosphorylation augments isometric twitch force during moderate, but not severe, levels of fatigue.     

Stretch activation and myosin heavy chain isoforms of rat,rabbit and human skeletal muscle fibres

The underlying mechanism of stretch-induced delayed force increase (stretch activation) of activated muscles is unknown. To assess the molecular correlate of this phenomenon, we measured stretch activation of single, Ca2+-activated skinned muscle fibres from rat, rabbit and the human and analysed their myosin heavy chain complement by SDS gradient gel electrophoresis. Stretch activation kinetics was found to be closely correlated with the myosin heavy chain isoform complement (I, IIa, IId/x and IIb). In hybrid fibres containing two myosin heavy chain isoforms (especially IId and IIb), the kinetics of stretch activation depended on the percentage distribution of the two isoforms. Muscle fibres of the same type but originating from different mammalian species exhibited similar kinetics of stretch activation. Considering the differing unloaded shortening velocities of these fibres, the time-limiting factors for stretch activation and unloaded shortening velocity appear not to be the same. The stretch activation kinetics of the fibre types IIB, IID and IIA more likely seemed to follow a Normal Gaussian distribution than that of type I fibres. Several type I fibres had extraordinarily slow kinetics. This observation corroborates biochemical data indicating the possible existence of more than one slow myosin heavy chain isoform     

De-phosphorylation of MyoD is linking nerve-evoked activity to fast myosin heavy chain expression in rodent adult skeletal muscle

Elucidating the molecular pathways linking electrical activity to gene expression is necessary for understanding the effects of exercise on muscle. Fast muscles express higher levels of MyoD and lower levels of myogenin than slow muscles, and we have previously linked myogenin to expression of oxidative enzymes. We here report that in slow muscles, compared with fast, 6 times as much of the MyoD is in an inactive form phosphorylated at T115. In fast muscles, 10 h of slow electrical stimulation had no effect on the total MyoD protein level, but the fraction of phosphorylated MyoD was increased 4-fold. Longer stimulation also decreased the total level of MyoD mRNA and protein, while the level of myogenin protein was increased. Fast patterned stimulation did not have any of these effects. Overexpression of wild type MyoD had variable effects in active slow muscles, but increased expression of fast myosin heavy chain in denervated muscles. In normally active soleus muscles, MyoD mutated at T115 (but not at S200) increased the number of fibres containing fast myosin from 50% to 85% in mice and from 13% to 62% in rats. These data establish de-phosphorylated active MyoD as a link between the pattern of electrical activity and fast fibre type in adult muscles.     

Effect of ageing on ultrastructure of slow and fast skeletal muscle tendon in rabbit Achilles tendons

This reports presents the changing morphological characteristics of collagen and fibroblasts in the soleus and gastrocnemius muscle tendon of female Japanese white rabbits with ageing. The fibroblasts decreased in number per 37 μm² with ageing in each group, and their morphology became longer and more slender through ageing. The mean fibril area and diameter of the collagen fibrils of soleus muscle tendon (SMT) and lateral gastrocnemius muscle tendon (GMT) in 8- to 10-month old rabbits were significantly higher than those of 3-wk-old rabbits during growth (P < 0.01). The mean area and diameter of collagen fibrils of SMT and GMT decreased during senescence: the values for 4- to 5-yr-old rabbits were lower than those for 8- to 10-month-old rabbits, but the difference was not significant. Statistically significant differences in fibril area and diameter between the SMT and GMT were not found during ageing. The number of thick fibrils increased during growth, but decreased in senescence. There were more thin fibrils (30–60 nm) in the 3-wk-old rabbits than in the 8- to 10-month old and 4 to 5-yr-old groups, and the large-diameter collagen (300–360 nm) was more abundant in the 8- to 10-month-old group than in the 3-wk-old and 4- to 5-yr-old groups. Differences in fibril size between slow and fast muscle tendons were not observed during ageing.     

Repositioning of fast and slow skeletal muscle

Summary The origins of the rat fast plantaris and slow soleus muscles were surgically reversed and their fibre types and contractile properties examinedin vitro up to 12 weeks post surgery. Muscles in which the origins had been severed and then immediately sutured back in place served as one control group. Unoperated animals served as a second control group. As compared to these groups, no significant differences in the histochemical or mechanical properties of the repositioned muscles were detected. Under the conditions of the experiments, no evidence was obtained to indicate that the intrinsic properties of a muscle could be altered by changing its site of origin and thus its functional environment.     

Changes in muscle fibre type, muscle mass and IGF-I gene expression in rabbit skeletal muscle subjected to stretch

The relationship between IGF-I and changes in muscle fibre phenotype in response to 6 d of stretch or disuse of the lower limb muscles of the rabbit was studied by combining in situ hybridisation and immunohistochemistry procedures. Passive stretch by plaster cast immobilisation of the muscle in its lengthened position not only induced an increase in IGF-I mRNA expression within the individual muscle fibres but also an increase in the percentage of fibres expressing neonatal and slow myosin. This change in phenotype was also found to be accompanied by a rapid and marked increase of muscle mass, total RNA content as well as IGF-I gene expression. In contrast, IGF-I appears not to be involved in muscle atrophy induced by immobilisation in the shortened position and the inactivity which results from this procedure. The level of increase in expression of IGF-I mRNA varied from fibre to fibre. By using adjacent serial sections, the fibres which expressed IGF-I mRNA at the highest levels were identified as expressing neonatal and the slow type 1 myosin. These data suggest that the expression of IGF-I within individual muscle fibres is correlated not only with hypertrophy but also with the muscle phenotypic adaptation that results from stretch and overload.