Rabbit Soleus Research Articles

Shortening velocity in single fibers from adult rabbit soleus muscles is correlated with myosin heavy chain composition.

Extensive variations exist in the heavy and light chain components of myosin in vertebrate striated muscles. In the present study, we have characterized a specific contractile property, velocity of shortening, and protein subunit composition of single fibers from adult rabbit soleus muscles. Maximum velocity of shortening (Vmax) was measured using the slack test method, and the myosin composition of these same fibers was determined using an ultrasensitive sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. While most fibers were found to have velocities between 0.5 and 1.0 muscle length/s, several had velocities distributed between 1.33 and 2.99 muscle length/s. The fibers in the slower group had myosin subunits that were solely of the slow type; however, those in the faster group contained both fast and slow heavy chains and light chains. The velocity of shortening measured in fibers having both myosin types was highly correlated with the myosin heavy chain composition, with velocity increasing as the proportion of fast-type heavy chain increased. Variations in light chain composition, particularly fast and slow myosin light chain 1, appeared to occur independently of the variations in heavy chain composition, suggesting that some myosin molecules consist of mixtures of slow- and fast-type subunits.

Location of C-protein, H-protein and X-protein in rabbit skeletal muscle fibre types.

The locations of C-protein, H-protein and X-protein in rabbit psoas, plantaris and soleus muscles have been investigated with fluorescently tagged specific antibodies. Two systems have been examined: isolated myofibrils allowed the locations of these proteins within the sarcomere to be determined, while cryosections allowed a comparison of the amounts of these proteins between different types of fibre in the three muscles. Using antibody-labelled cryosections, we find that the amounts of each of these proteins depends closely on the fibre type. In all the muscles studied, C-protein is present in the largest amounts in fast white and fast intermediate fibres and is absent from slow red fibres, while X-protein is absent from fast white fibres and is present in the largest amounts in fast and slow red fibres. In psoas muscle, H-protein is present in the largest amounts in fast white fibres and is absent in fast and slow red fibres. In plantaris muscle, however, H-protein is absent from fast white fibres but occurs in some slow red fibres. All psoas myofibrils label with anti-C and anti-H and a minority label with anti-X. In each case the pattern of labelling is a zone in each half of the A-band. Measured across the middle of the A-band, the zones for H-protein are much closer together than for C-protein; the centre-to-centre spacings are 0.35 micron for anti-H and 0.64 micron for anti-C. The fluorescent zones for X-protein are slightly but significantly closer (0.52 micron) than those for C-protein. All soleus myofibrils label with anti-X but the centre-to-centre spacing was greater (0.67 micron). With plantaris myofibrils, where labelling occurs with anti-C or anti-H, the spacings resemble those in psoas myofibrils, but with anti-X the spacing resembles that in soleus myofibrils. The spacing of the fluorescent zones in an A-band, whether produced by anti-C, anti-X or anti-H does not vary with sarcomere length. We conclude that X-protein and H-protein, like C-protein, are thick filament components. With both fibres and myofibrils, there is no simple relationship between the amount of X-protein and the amount of C-protein. Many fast intermediate fibres in psoas and plantaris muscle label as strongly with anti-C as do fast white fibres but also label as strongly with anti-X as do fast and slow red fibres.(ABSTRACT TRUNCATED AT 400 WORDS)

Force response to width and length perturbations in compressed skinned skeletal muscle fibers.

Previous studies have shown that radial compression of calcium-activated skinned skeletal muscle fibers, with attendant reduction of filament lattice spacing, reduces isometric force generation. In relaxed skinned fibers, radial compression produces a marked increase in axial elastic modulus, and the response to a small amplitude length perturbation resembles that of a muscle in rigor. We interpret these results as indicating that radial compression of the myofilament lattice produces "hindered" cross-bridges which are load bearing but not force generating. The experiments reported here were designed to study the effect(s) of "hindered" cross-bridges on both the time course of isometric force responses following Ca2+ activation and fiber width and length perturbations. The experiments were carried out at room temperature on radially compressed skinned single rabbit soleus fibers. Force development following step-wise Ca2+ activation and step-wise changes of fiber width was "slow" (60-90 sec) compared to that in normal width fibers (approximately 1 sec), and could be approximated by a single exponential curve. Force redevelopment following a length release in compressed fibers was both more rapid and more complicated than force development following activation and width steps, and required a double exponential curve for an adequate description. The results are consistent with the notion that hindered cross-bridges form as a result of lattice compression, and that the hindered bridges affect the force responses following width and length perturbations.

Tension and ATPase rate in steady-state contractions of rabbit soleus fiber segments.

Steady-state isometric tension and ATPase were studied in hyperpermeable segments of single muscle fibers from rabbit soleus muscle at 22 degrees C. The ATPase activity was due to actomyosin. The ratio of fiber ATPase to tension was used as an index of steady-state cross-bridge kinetics. Increasing the calcium ion concentration from pCa 8 to pCa 5 activated both tension and ATPase. The maximal tension was 1.35 +/- 0.07 kg/cm2. The maximal ATPase was 1.05 +/- 0.13 mumol X g-1. s-1 at pCa 5.2. ATPase activity increased with tension, such that the ratio of ATPase to tension remained constant at all calcium concentrations. In the absence of calcium, increasing the concentration of MgATP from 1 to 7 X 10(-7) M increased tension from zero to a maximum of 0.46 +/- 0.03 kg/cm2. Increasing MgATP concentration further to 1 X 10(-6) M inhibited tension. In the phase of rising tension, ATPase increased proportionally to tension, to 0.11 +/- 0.01 mumol X g-1 X s-1 at maximum tension. However, the ratio of ATPase to tension on the rising phase had a value only one-third of that seen with calcium-activated tension. Thus, low substrate concentrations, but not low calcium ion concentrations, influence cross-bridge kinetics under steady-state isometric conditions, possibly by an increase in the tension-time product during a cross-bridge cycle.

The relation of neuromuscular synapse elimination to spinal position of rabbit and rat soleus motoneurones.

We have examined recent claims that neuromuscular synapse elimination occurs preferentially among motoneurones from the more rostral of the two spinal roots contributing to the soleus muscle of the rat. The number of synapses made by individual motoneurones contributing to the soleus muscles of the rabbit and rat were estimated at different stages of post-natal development. The assay was based on measurements of twitch tensions in response to stimulation of individual motor axons in ventral root filaments. Contrary to the previous claims, we found that synapses were lost by motoneurones passing through all contributing spinal roots in both the rabbit and rat. Furthermore, in both species there was little or no difference in the extent of synapse elimination associated with the various spinal roots.

Ipsi- and contralateral fibre transformations by cross-reinnervation. A principle of symmetry.

Cross-reinnervation of rabbit soleus muscle by the peroneal nerve induces a 90% transformation of slow into fast fibres. These changes are reflected in corresponding transformations of the enzyme activity pattern of energy metabolism, the isozyme pattern of lactate dehydrogenase and, in confirmation of previous results (Srihari et al. 1981), transitions from a slow to a fast type myosin light chain pattern. The transformation process appears to be complete after 6 months. Similar changes, although less extensive are also found in the soleus muscle of the contralateral leg. Fibre type transitions in the contralateral muscle are not accompanied by fibre type grouping, as seen in the cross-reinnervated muscle and therefore these changes appear to result from a transformation of the motor units themselves. This phenomenon is interpreted as a compensatory process in maintaining symmetry within the neuromotor system.

The effect of cross-innervation on the tropomyosin composition of rabbit skeletal muscle

Soleus, semitendinosus and crureus muscles of the rabbit were found to contain α- and β-tropomyosin subunits and additional forms that have been provisionally designated γ and δ. Extensor digitorum longus and psoas muscles contained only α and β subunits, the relative proportions of which varied between single fibres of psoas muscle. On cross-innervation of rabbit soleus and extensor digitorum longus muscles, the fraction of the total tropomyosin present as the β subunit remained constant. The relative proportions of α, γ and δ subunits changed as would be expected from the change in speed that occurred.

Changes in transcriptional activity of chronically stimulated fast twitch muscle

mRNAs extracted from rabbit soleus, normal and 28-day, indirectly stimulated tibialis anterior muscles were translated in an in vitro system. Analysis for translation products by 2-dimensional electrophoresis showed fast myosin light chains in tibialis anterior, and slow myosin light chains in soleus muscle. The stoichiometry of the in vitro translated light chain varies from that seen in normal fast and slow twitch muscles. The stimulated muscle contained mRNA coding, both for fast and slow myosin light chains, although the pattern of slow myosin light chains appears not to be complete at this point of time of the transformation process.

α- and β-tropomyosin in typed single fibers of human skeletal muscle

Tropomyosin, in association with the troponin, is involved in the calcium-dependent regulation of act inmyosin interaction [ 1 ]. In skeletal muscles two major forms have been detected. In the rabbit, both of them possess 284 amino acid residues, 39 of which are different [2]. In spite of this similarity, their mobilities on SDS-polyacrylamide gels clearly differ. By convention, the faster migrating form is called c~-t ropomyosin, and the slower form 13-tropomyosin [3]. Minor forms have been described for c~and for /3-tropomyosin, differing in their primary sequences [2,4]. In cat [5] and chicken [6] skeletal nmscle, different c~-forlns associated with fast and slow skeletal muscle have been found. Furthermore, skeletal muscle tropomyosins can be phosphorylated, resulting in different electrophoretic mobilities [6]. It has been suggested that in mammals (and in particular in man), c~-tropomyosin is confined to type lI-fibers and ~3-tropomyosin to type I-fibers [7,8]. This contradicts the above-mentioned results from cat muscle [5]. Furthermore, in rabbit soleus muscle containing only few type lI fibers [9], both c~and 13-forms are present in roughly equal amounts [ 10]. Taking advantage of recently developed techniques for the analysis of the protein composition of typed single muscle fibers [ 11 ], we demonstrate in this study that in human skeletal muscle c~and 13-tropomyosins are present in about equal amounts in type I, IIA and IIB fibers. We also present evidence that the c~and H-forms display fiber type-specific characteristics. For this purpose, a new, two-dimensional peptide mapping

C-protein from rabbit soleus (red) muscle

A new form of skeletal-muscle C-protein has been isolated from rabbit soleus (red) muscle. This new form of C-protein has been purified to homogeneity by a procedure similar to that used to purify C-protein from white skeletal muscle. In soleus muscle, only this new form of C-protein could be detected, whereas in psoas (white) muscle, only the previously identified form of C-protein was detected. The content of C-protein in rabbit soleus muscle is comparable with that found in psoas muscle. Other rabbit skeletal muscles composed of a mixture of fibre types contained at least two forms of C-protein. C-Protein derived from red skeletal muscle bound to myosin isolated from either red or white tissue, with maximum binding occurring at a ratio of approximately 13 microgram of red C-protein/100 microgram of myosin. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that C-protein isolated from red skeletal muscle has a molecular weight approx. 7% greater than that of C-protein isolated from white skeletal muscle. The amino acid content of both forms of C-protein was similar but major differences in the mol % of isoleucine and threonine were found. Antiserum against C-protein from white rabbit skeletal muscle formed a single precipitin line with rabbit C-protein on double in agar. This antiserum did not form a precipitin line when diffused against red C-protein from rabbit skeletal muscle. Also, this antiserum bound specifically to the A-band region of myofibrils isolated from psoas (white) muscle, but it did not bind to myofibrils prepared from soleus (red) muscle.

The effect of sarcomere non-uniformity on the sarcomere length-tension relationship of skinned fibers.

It has proved difficult to activate skinned muscle fibers to produce high tension (3 kg/cm2 level) without loss of clear striations. A new method was developed which permits high tension production in skinned muscle fibers while retaining clear striations. Clear striations allow reliable measurement of the sarcomere lengths during contraction by microscopy and diffractometry. The method is to increase the Ca++ concentration of the bathing solution very gradually over a time period of 5 to 10 minutes. Once the skinned fiber is conditioned by this slow activation, subsequent contractions can be elicited by ordinary quick activations without loss of striations. When the experiments are carried out with careful controls for the uniformity of the sarcomere length distribution along the entire length of the fiber, contractions are highly repeatable. Using the new method and stringent quality control of fibers, the sarcomere length-isometric tension relationship of skinned rabbit soleus fibers was obtained. The results differ from those previously obtained by conventional activation methods in that tension increases with sarcomere length not only at low (pCa = 5.8), but also at high (pCa = 5.2), calcium concentration.

Proteolytic digestion patterns of myosin from new-born and adult fast, slow, and mixed skeletal muscle

1. 1. Fragments produced by proteolysis of various skeletal muscle myosins with trypsin have been investigated by sodium dodecylsulfate (SDS) gel electrophoresis. 2. 2. Tryptic digestion of new-born rabbit soleus muscle and new-born rabbit extensor digitorum longus muscle (EDL) myosin revealed a peptide pattern very similar in both cases, but distinct from the pattern obtained after digestion of corresponding adult rabbit muscle myosins. 3. 3. The digestion pattern of the adult rabbit diaphragm is identical with that obtained by digestion of mixture of adult EDL and soleus muscle myosins.

Thermal denaturation of proteins in Post rigor muscle tissue as studied by differential scanning calorimetry

AbstractPost rigor bovine M. semimembranosus was analysed by differential scanning calorimetry (d.s.c.). After extractive removal of sarcoplasmic proteins, subsequent pH adjustment and manual connective tissue removal, d.s.c. yielded reproducible thermograms which permitted investigation of the individual major myofibrillar proteins in various pH and salt environments without prior isolation. The positions of two major peaks, interpreted as myosin transitions, proved to be strongly pH dependent. At pH 5.4, the peak maxima occurred at 58 and 65°C, respectively, at a heating rate of 10°C min‐1. Above pH 6.5 their order of denaturation was reversed. In the pH range 5.4–6.5 the peak ascribed to actin had its maximum near 80°C in intact muscle. Above this pH range it was displaced to lower temperature. The thermal stability of actin was studied after treatment of the muscle tissue with different salt solutions. At equal ionic strengths (μ = 0.15) at pH 5.5, calcium chloride and sodium chloride caused 6.5°C and 4°C displacement to lower temperature, respectively. The thermograms of bovine semimembranosus muscle were compared to those of two red and two white muscle types (bovine cardiac and rabbit soleus muscles, chicken breast and rabbit semimembranosus muscles, respectively) at two pH levels. Greater myosin differences were found between red and white muscles than between muscles from different animal species. All muscles gave similar actin transitions, with exception of the heart muscle where the actin peak appeared at 3 °C lower temperature. The necessity of a strict pH control in order to obtain reproducible muscle thermograms is demonstrated.

Effects of halothane on functionally skinned rabbit soleus muscle fibers: a correlation between tension transient and 45Ca release.

The mechanism(s) involved in the halothane-induced increase in skeletal muscle contraction was studied using functionally skinned soleus muscle fibers from rabbits: For the tension study, single functionally skinned fibers were individually mounted on two pairs of forceps, with one end attached to a photodiode tension transducer. Ca2+-activated tension development of the contractile proteins, and Ca2+ uptake and release from the sarcoplasmic reticulum (SR) using caffeine-induced tension transients were studied. To measure the amount of calcium, skinned fibers at 0.1 g/ml were used and 0.075 μCi45Ca/ml was spiked in the solution 3 (pCa 6.5 and 1 mM [EGTA]) which promoted rapid loading of Ca2+. Halothane (1–3%) did not change the [Ca2+]-tension relationship; 2 and 3% halothane reduced the maximum Ca2+-activated tension by 6–7%. Halothane (1–3%) added to the solution 3, reduced45Ca uptake by 3, 22 and 23%; however, the subsequent caffeine-induced tension transient and45Ca release were increased by 10–40%. During the release phase only halothane increased both caffeine-induced tension transient and45Ca release by 20–60%. The effects of halothane on the tension transient and on the45Ca release were comparable. There was no dose-response relationship to the effects of halothane on the above parameters. It is concluded that halothane affects the SR by increasing its membrane permeability to Ca2+, resulting in an increase in myoplasmic [Ca2+] and thus in the twitch tension in skeletal muscle.

Associated changes in Ca2+ and Sr2+ activation properties and fiber proteins in cross-reinnervated rabbit soleus.

Changes in the Ca2+ and Sr2+ activation properties of functionally skinned slow-twitch soleus fibers were measured and compared with those of normal fast-twitch extensor digitorum longus (EDL) following cross-reinnervation of soleus with the nerve to EDL. Most of the fibers showed either complete transformation of activtion properties (66%) or remained unchanged (34%). The change in sensitivity to divalent cations was correlated with changes in the proteins present in fibers pooled on the basis of their activation properties. The banding patterns of the 35,000- and 37,500-dalton proteins (tropomyosin and troponin T) in cross-reinnervated soleus were correspondingly transformed to those of normal EDL. Slow and fast myosin light chains were present in the pooled cross-reinnervated fibers. Fiber distributions based on activation properties were confirmed by histochemical features. For the first time it has been demonstrated that cross-reinnervation produced changes in the activation properties of soleus fibers and associated changes in the regulatory proteins measured.

Nonparallel isometric tension response of rabbit soleus skinned muscle fibers to magnesium adenosine triphosphate and magnesium inosine triphosphate.

The isometric tension response of single "skinned' rabbit soleus muscle fibers to MgATP and McITP in the absence of calcium was studied. [MgATP] or [MgITP] was varied in solutions of ionic strength 0.30 and temperature 20 degrees C. Steady-state tension that developed in MgATP or MgITP solutions was a biphasic bell-shaped function of log [MgATP] or log [MgITP] which increased from zero to maximum tension and then declined again to zero. Analysis of the data showed that, under comparable ionic conditions, percent tension vs. log [MgATP] and percent tension vs. log [MgITP] curves are not parallel. Instead, the percent tension vs. log [MgITP] curve is much broader. Additionally, under comparable ionic conditions maximum tension in MgITP solutions was higher than in MgATP solutions. In addition, in MgATP solutions, pH, [K+], and excess ATP were varied. Raising pH from 7 to 8, [K+] from 46 mM to 200 mM, or decreasing excess ATP from 2 to 0.5 mM all increased maximum tension. None of these factors, however, influenced the shape or position of the percent tension vs. log [MgATP] curve.

The effects of tenotomy upon the contraction characteristics of motor units in rabbit soleus muscle.

1. Isometric contractions of tenotomized rabbit solei have been compared with a group of unoperated control muscles. A large decrease in the tension developed by the tenotomized muscle was accompanied by a slight shortening of the twitch contraction time, and a larger, progressive, reduction in the time to half relaxation.2. A comparison of motor units obtained from muscles which had been tenotomized for six weeks with those of the control muscles showed a large reduction in the range of contraction and half relaxation times of the units from the tenotomized muscles.3. The mean motor unit tension (expressed as a percentage of the whole muscle tension) was similar for both those units from the control muscles and those from muscles which had been tenotomized for six weeks, indicating a uniform atrophy of motor units within the tenotomized muscles.4. It was concluded that the change in the pattern of motor unit contraction times was not the result of a process of differential atrophy favouring the preservation of the faster contracting motor units.5. A correlation between axon conduction velocity and both the speed of contraction and the size (tension) of the motor units was demonstrated in the control muscles. Following tenotomy the relationship between axon conduction velocity and motor unit tension was lost.

Differential, direct effects of H+ on Ca2+ -activated force of skinned fibers from the soleus, cardiac and adductor magnus muscles of rabbits.

The effect of acidosis on Ca2+-activated force generation was studied in rabbit soleus, left ventricular, and adductor magnus muscles. Fibers were skinned (sarcolemma peeled off or mechanico-chemically disrupted) to facilitate direct manipulation and standardization of their intracellular ionic milieus according to bathing solution composition. Skinned single skeletal and small bundles of cardiac fibers were mounted in a photodiode force transducer and activated by immersion in buffered-Ca2+ bathing solutions. The magnitude of steady state isometric force at each [Ca2+] was determined at pH 7.0 and 6.5 (paired data) at both 1 mM and 10 mM Mg2+ in order to detect artifacts of errors in calculated [Ca2+]. All bathing solutions contained: 7 mM total EGTA [ethyleneglycol-bis-(β-amino-ethylether)-N,N′ tetra-acetic acid], 70 mM (Na++K+), 2 mM MgATP2− (Mg adenosine triphosphate), 15 mM CP2− (creatine phosphate), 15 units/ml CPK (creatine phosphokinase), imidazole (adjusted ionic strength to 0.15 M), and propionate anion at 23±1° C. Maximum tensions were similar at both [Mg2+]s but less at pH 6.5 than at pH 7.0, with the following order of mean magnitude of acidotic depression adductor>cardiac>soleus. The proportionately greater acidotic depression of submaximum (relative to maximum) forces that occurred only at 1 mM Mg2+ (cardiac>adductor>soleus) implicates acidotic depression of Ca2+-activated force as a major cause of decreased cardiac contractility.

Light chains from slow-twitch muscle myosin.

Myosin light chains have been isolated from slow-twitch soleus muscles of rabbit and cat. Two chemically related light chains of molecular weight about 22 000 have been identified from their thiol sequences in each species, and these have been further characterized by amino acid analysis and peptide mapping studies. These light chains are related to the alkali light chains of rabbit fast-twitch muscles and to the larger cardiac light chain from bovine heart muscle. The presence of two chemically related but phenotypically distinct light chains within single muscles suggests the presence of myosin isoenzyme or that the myosin molecule has a different light chain associated with each subfragment-1 head. Although the stoichiometry of these two light chains had not been determined, the former of these two conclusions is favoured by analogy with experiments on fast-twitch myosins. In addition to these related light chains, soleus muscle myosin, like fast-twitch myosins, contains a third light chain of about 19 000 molecular weight. Unlike the corresponding light chain of rabbit fast-twitch myosin, this 19 000-Mr light chain contrains no cysteine residues. The distribution of thiol peptides together with the characteristic mobilities of these light chains on polyacrylamide gels in the presence of sodium dodecyl sulphate provides a 'fingerprint' of myosins from different muscle types. This has been used to look for the presence of fast-twitch myosin in soleus muscle, the results showing both rabbit and cat soleus muscles are homogeneous in their myosin type within the sensitivity of detection of the methods. These techniques have also been used to confirm the reciprocal transformation of light chains in myosins isolated from cross-reinnervated muscles. Finally the relationships between these criteria for different myosin types and histochemical procedures for muscle fibre typing are discussed.

Phosphorylation of the light-chain components of myosin from cardiac and red skeletal muscles.

1. The light-chain components of myosin from cardiac muscle (19000 and 27000 daltons) and of rabbit soleus and crureus muscles (19000, 27000 and 29000 daltons) were characterized. 2. The 19000-dalton components in carciac- and red-skeletal-muscle myosins were spontaneously modified to a component of slightly higher net negative charge. 3. The 19000-dalton component in cardiac and red skeletal muscles and their modified forms were phosphorylated by myosin light-chain kinase. 4. Evidence was obtained for the presence of myosin light-chain kinase in cardiac and red skeletal muscles. 5. Myosin light-chain kinase catalysed the phosphorylation of the whole light-chain fraction from white and red skeletal muscle at similar rates. The light-chain fraction of cardiac-muscle myosin was phosphorylated at a significantly lower rate. 6. The light-chain components of cardiac-muscle myosin and their phosphorylated froms were separated by ion-exchange chromatography and their amino acid compositions determined.