Myogenic and neurogenic regulation of myosin gene expression in cat jaw-closing muscles regenerating in fast and slow limb muscle beds

Summary Immunocytochemical techniques were used to study changes in myosin gene expression during the regeneration of the cat posterior temporalis muscle transplanted into the bed of either the fast extensor digitorum longus (EDL) or the slow soleus muscle. Strips of the posterior temporalis, a homogeneously superfast muscle, were treated with Marcaine and then transplanted into limb muscle beds which had been completely cleared of host muscle fibres. The regenerates were examined 6 to 224 days after surgery. Early regenerates in both muscle beds reacted with antibodies against the heavy chain of foetal, slow and superfast myosins, but not with antibodies against fast myosin. In the long-term, regenerates innervated by the EDL nerve expressed only superfast myosin whereas in the regenerates innervated by the soleus nerve most fibres expressed only slow myosin and only a few fibres reacted exclusively with the anti-superfast myosin antibody even after 210 days. In contrast, EDL and soleus muscles regenerating in their own beds expressed foetal, slow and fast myosin, but did not express superfast myosin. The isometric contraction times of the various types of regenerates reflected the types of myosin synthesized. It is concluded that jaw and limb muscle cells exist as two distinct allotypes, each having a distinct repertoire for the expression of adult isomyosins, and that within that repertoire isomyosin gene expression can be modulated by the nerve. Thus, myosin gene expression in skeletal muscle fibres is regulated by both myogenic and neurogenic mechanisms.     

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.     

Distinct myosin heavy chain isoform transitions in developing slow and fast cat hindlimb muscles

The expression of myosin heavy chain (MHC) isoforms leading to adult fiber phenotypes in the tibialis anterior (TA) and soleus muscles of the cat were investigated from embryonic day 35 to 1 year after birth. Electrophoresis and immunoblotting of myofibrils demonstrated the expression of 5 different MHC isoforms, i.e. I, IIa, IIx, embryonic, and neonatal, during development. Based on electrophoresis, the adult-like MHC composition of the soleus and TA were not observed until postnatal day 40 (P40) and 120 (P120), respectively. In contrast, immunohistochemical analyses revealed that the adult-like fiber phenotype composition was attained much later (P120) in the soleus. The existence of multiple MHC isoforms in individual fibers suggested that transitions occurred until P120 in both muscles. Adult type I fibers were first observed at P1. Adult IIA fibers were first observed at P30 in the TA and P40 in the soleus. IIX fibers were not identified until P40 in the TA. The transition to the predominantly slow phenotype of the soleus involved a gradual loss of embryonic and fast isoforms accompanied by an accumulation of slow MHC. In contrast, the expression of slow and fast MHC in the fast TA muscle was relatively unchanged throughout development. These results show that the establishment of a given MHC-based fiber phenotype varies significantly between slow and fast muscles in the kitten.     

Enzymic properties of myosin in fast and slow twitch muscles of the cat following cross-innervation

1. Following cross-innervation of the cat flexor digitorum longus muscle (F.D.L., fast-twitch) and soleus muscle (slow-twitch) the isometric twitch speed of the F.D.L. muscle decreases while the contraction time of soleus increases. The tetanus-twitch ratio of soleus is also increased.2. Myosin or myofibrils from normal or self-reinnervated fast-twitch muscles of the cat split adenosine triphosphate (ATP) more rapidly than myosin or myofibrils from slow twitch muscles.3. Following cross-innervation the myosin and myofibrillar ATPase activity of flexor digitorum longus muscle falls, to become nearly equal to that of normal or self-reinnervated soleus muscle. The ATPase activity of cross-innervated soleus muscle increases only slightly.     

Isometric endurance in fast and slow muscles in the cat.

By use of a method of sequential stimulation described previously, the ability to sustain tensions of 3--100% of the initial strength (maximum isometric tension that could be developed in the fresh muscle) to fatigue (endurance) was assessed in the soleus (slow-twitch), medial gastrocnemius (mixed, fast-twitch), and plantaris (mainly fast-twitch) muscles. For all fractions of the initial strength the endurance was longest in soleus and shortest in plantaris. However, although plantaris fatigued at any tension examined, soleus could maintain a tension of up to 30% of its initial strength indefinitely with no sign of fatigue. Part of the difference in endurance between these two muscles could be accounted for in terms of blood flow because arterial occlusion sharply reduced the endurance of soleus but had only a small effect on endurance in plantaris. However, even with an occluded circulation, soleus still had substantially longer endurance than either medial gastrocnemius or plantaris. The origin of fatigue in any of the three muscles examined appeared to be in the muscle; there was no evidence of failure of transmission at the neuromuscular junction.     

Fibre types in extraocular muscles: a new myosin isoform in the fast fibres

Summary We report on the existence of a myosin heavy chain (MHC) isoform with unique structural properties in extraocular (EO) muscles. Differences in MHC composition are apparent using a polyclonal antibody prepared against myosin isolated from bovine EO muscle myosin. In enzyme immunoassays and western blotting experiments, this anti-EO myosin antibody reacted specifically with the heavy chains of EO muscle myosin and not with the heavy chains of other myosins. The distribution of this new MHC isoform in the globe rotating muscles from different mammalian species was analysed using a panel of specific anti-myosin antibodies and comparing the histochemical myosin ATPase profile of muscle fibres with their isomyosin content. Most fibres which display a type 2 ATPase reaction pattern were selectively labelled by anti-EO antibodies. A few type 2 fibres were found to react with both anti-EO and anti-2A myosin antibodies and others, located almost exclusively in the orbital layers, reacted with anti-foetals as well as anti-EO antibodies.The presence of a distinct form of myosin in EO muscle fibres is probably related to the particular functional characteristics of these muscles, which are known to be exceptionally fast-contracting but display a very low tension output.     

Energy cost of submaximal isometric contractions in cat fast and slow twitch muscles

The purpose of the present investigation was to compare the net energy cost incurred by cat soleus (slow twitch muscle) and medial gastrocnemius (predominantly fast twitch muscle) muscles for isometric contractions. For this, a computer-controlled sequential stimulation system was employed that enabled fused isometric contractions at frequencies of motor unit discharge within the normal physiological range. This allowed submaximal isometric contractions to be maintained at tensions of 10%, 25%, 50% and 75% of the initial strength of each muscle (tetanic tension of the unfatigued muscle determined at the beginning of each experiment). Total net energy cost was greater for the gastrocnemius than for the soleus muscle at each tension studied. For both muscles, the metabolism shifted toward anaerobic pathways at higher contraction tensions. But in comparison to the soleus muscle, the gastrocnemius muscle consistently had a greater percentage of its total net energy cost provided by anaerobic glycolysis rather than aerobic metabolism; for the gastrocnemius and soleus muscles the percent of the total metabolism from anaerobic pathways was 74% and 18% during the 10% contraction, and 96% and 84% during the 75% contraction for the medial gastrocnemius and soleus muscles respectively.     

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.     

Enzymic and physiological studies on normal and disused developing fast and slow cat muscles

1. Contraction time (CT), (1/2) relaxation time ((1/2) RT), maximum twitch-tetanus tensions and the activity of two glycolytic enzymes (aldolase, pyruvic kinase) and two oxidative enzymes (malic and isocitric dehydrogenase) was studied in anterior tibialis (AT) and soleus (SOL) muscles from birth to 18 weeks of age.2. One hind leg of each of six kittens was casted at birth and the above parameters were examined in both casted and contralateral AT and SOL at 18 weeks of age.3. Differentiation of the contraction properties in normal developing fast muscle (AT) was closely paralleled by a marked elevation in glycolytic enzyme activity. Oxidative enzymic activity in AT and SOL was relatively unchanged from birth to 18 weeks of age, as was glycolytic activity in SOL.4. Disuse, due to immobilization, produced atrophy in AT and SOL muscles at 18 weeks of age. The CT of the casted SOL was slightly quicker than normal while the CT of the AT was unchanged. The glycolytic and oxidative enzymic activity in both casted muscles was generally unchanged but the contralateral AT had higher glycolytic activity than normal muscles.     

Isometric contractions of motor units in self-reinnervated fast and slow twitch muscles of the cat

1. Isometric contractions of motor units have been studied in self-reinnervated soleus and flexor digitorum longus muscles, the motor nerves of which had been transected 6 months earlier.2. The distributions of motor unit tetanic tensions in both muscles were skewed towards large values and showed greater variance than normal: there were motor units which produced more tension and others which developed less tension than in control muscles. Mean tensions of motor units are compared with those of the controls.3. The variance of times to peak of motor units in both muscles was significantly smaller than normal.4. Conduction velocities of axons were slower than normal above the point of nerve section. A number of axons did not functionally cross this point and those that did conducted slowly below the neuroma.5. Many of the correlations seen in normal muscle between axon conduction velocity, motor unit tetanic tension, twitch time to peak and ratio of twitch to tetanic tension were demonstrated in reinnervated muscle.6. We suggest that the pattern of motor unit organization in self-reinnervated muscle can in part be due to random regrowth of axons but is largely produced by specific processes related to axon size which are not identical in fast and slow muscle.     

Influence of fast and slow alkali myosin light chain isoforms on the kinetics of stretch-induced force transients of fast-twitch type IIA fibres of rat

This study contributes to understand the physiological role of slow myosin light chain isoforms in fast-twitch type IIA fibres of skeletal muscle. These isoforms are often attached to the myosin necks of rat type IIA fibres, whereby the slow alkali myosin light chain isoform MLC1s is much more frequent and abundant than the slow regulatory myosin light chain isoform MLC2s. In the present study, single-skinned rat type IIA fibres were maximally Ca²⁺ activated and subjected to stepwise stretches for causing a perturbation of myosin head pulling cycles. From the time course of the resulting force transients, myosin head kinetics was deduced. Fibres containing MLC1s exhibited slower kinetics independently of the presence or absence of MLC2s. At the maximal MLC1s concentration of about 75%, the slowing was about 40%. The slowing effect of MLC1s is possibly due to differences in the myosin heavy chain binding sites of the fast and slow alkali MLC isoforms, which changes the rigidity of the myosin neck. Compared with the impact of myosin heavy chain isoforms in various fast-twitch fibre types, the influence of MLC1s on myosin head kinetics of type IIA fibres is much smaller. In conclusion, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the myosin head kinetics.     

Dynamic properties of fast and slow skeletal muscles in the cat and rat following cross-reinnervation.

1. Cross-reinnervation was performed between the slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles in the cat and the SOL and fast-twitch flexor hallucis longus (FHL) muscles in the rat. The isometric and force-velocity properties of the muscles were subsequently determined. 2. As a result of cross-reinnervation the dynamic properties of both slow- and fast-twitch muscles in the cat were considerably altered. The isometric twitch time to peak was 33-9 msec in the X-SOL compared with 84-7 msec in the N-SOL. The intrinsic speed of shortening and a/P0 values were 25-4 mum. sec-1 and 0-34 respectively in the X-SOL and 14-4 mum. sec-1 and 0-20 in the N-SOL. In the X-EDL the twitch time to peak was 52-6 msec compared with 23-4 msec in the N-EDL; the intrinsic speed of shortening and a/P0 values were 14-1 mum. sec-1 and 0-29 respectively, whereas they were 31-9 mum. sec-1 and 0-44 in the N-EDL. 3. In the rat X-SOL there was shortening of the isometric twitch time to peak from 39-5 msec in the N-SOL to 19-7 msec and an increase in the intrinsic speed of shortening from 17-7 mum. sec-1 to 32-3 mum. sec-1, although over most of the force-velocity curve the X-SOL values were only slightly greater than those for N-SOL. The force-velocity data for the X-SOL muscles could not be fitted by Hill's equation. 4. The isometric and force-velocity results of the X-SOL of the rat suggested that the muscle contained a significant proportion of slow fibres. An examination of the motor unit characteristics of the N-FHL showed the presence of approximately 14% small, slow units. 5. It is concluded that there are no fundamental differences between the rat and the cat with regard to the effect of cross-reinnervation on hind-limb muscles. However, these, and previously published results, suggest that there may be important differences between heterologous muscles in the degree of influence that their respective motor nerves can exert on an alien muscle.     

Fibre-type specific expression of fast and slow essential myosin light chain mRNAs in trained human skeletal muscles

The fibre-type specific expression patterns of fast and slow isoforms of essential (alkali) myosin light chains (ELC) was analysed in trained, untrained and pathological human muscles. Biopsies from m. vastus lateralis of moderately trained and untrained persons, as well as highly trained endurance and strength athletes were analysed, by in situ hybridization, for the expression of the `fast' ELC 1f/3f and the `slow' ELC 1sb. We wanted to investigate if changes in the fibre-type specific ELC mRNA pattern could be used as markers for training adaptation, especially, if the mRNA of the slow ELC 1sb isoform would appear in type IIA fibres as a result of endurance training (Baumann et al. 1987). We found the fast/slow ELC expression patterns in the fibre types to be remarkably stable. Physiological stress, even high training loads, did not affect it. No IIA fibres expressing ELC 1sb mRNA were found. They could be detected, however, in pathological muscle samples, where fast/slow ELC patterns not found in normal muscles were frequent. Our data suggest that in healthy muscles, only a subset of the theoretically possible combinations of myosin heavy and light chain isoforms are expressed at the level of their mRNAs.     

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.     

Actomyosin kinetics of pure fast and slow rat myosin isoforms studied by in vitro motility assay approach

An in vitro motility assay approach was used to investigate the mechanisms of the functional differences between myosin isoforms, by studying the effect of MgATP and MgADP on actin sliding velocity (V(f)) of pure slow and fast rat skeletal myosin at different temperatures. The value of V(f) depended on [MgATP] according to Michaelis-Menten kinetics, with an apparent constant (K(m)) of 54.2, 64.4 and 200 μm for the fast isoform and 18.6, 36.5 and 45.5 μM for the slow isoform at 20, 25 and 35°C, respectively. The presence of 2 mM MgADP decreased V(f) and yielded an inhibition constant (K(i)) of 377, 463 and 533 μM for the fast isoform at 20, 25 and 35°C, respectively, and 120 and 355 μM for the slow isoform at 25 and 35°C, respectively. The analysis of K(m) and K(i) suggested that slow and fast isoforms differ in the kinetics limiting V(f). Moreover, the higher sensitivity of the fast myosin isoform to a drop in [MgATP] is consistent with the higher fatigability of fast fibres than slow fibres. From the Michaelis-Menten relation in the absence of MgADP, we calculated the rate of actomyosin dissociation by MgATP (k(+ATP)) and the rate of MgADP release (k(-ADP)). We found values of k(+ATP) of 4.8 × 10(6), 6.5 × 10(6) and 6.6 × 10(6) M(-1) s(-1) for the fast isoform and 3.3 × 10(6), 2.9 × 10(6) and 6.7 × 10(6) M(-1) s(-1) for the slow isoform and values of k(-ADP) of 263, 420 and 1320 s(-1) for the fast isoform and 62, 107 and 306 s(-1) for the slow isoform at 20, 25 and 35°C, respectively. The results suggest that k(-ADP) could be the major determinant of functional differences between the fast and slow myosin isoforms at physiological temperatures.     

Differences in myosin composition between human oro-facial,masticatory and limb muscles: enzyme-, immunohisto-and biochemical studies

Summary Immunohistochemistry was used to determine the myosin composition of defined fibre types of three embryologically different adult muscles, the oro-facial, masseter and limb muscles. In addition, the myosin composition in whole muscle specimens was analysed with biochemical methods. Both similarities and differences between muscles in the content of myosin heavy chains and myosin light chains were found. Nevertheless, each muscle had its own distinct identity. Our results indicated the presence of a previously undetected fast myosin heavy chain isoform in the oro-facial type II fibre population, tentatively termed fast F. The masseter contained aberrant myosin isoforms, such as foetal myosin heavy chain and -cardiac myosin heavy chain and unique combinations of myosin heavy chain isoforms which were not found in the limb or oro-facial muscles. The type IM and IIC fibres coexpressed slow and fast A myosin heavy chains in the oro-facial and limb muscles but slow and a fast B like myosin heavy chain in the masseter. While single oro-facial and limb muscle fibres contained one or two myosin heavy chain types, single masseter fibres coexpressed up to four different myosin heavy chain isoforms. Describing the fibres according to their expression of myosin heavy chain isozymes, up to five fibre types could be distinguished in the oro-facial and limb muscles and eight in the masseter. Oro-facial and limb muscles expressed five myosin light chains, MLC_1S, MLC_2S, MLC_1F, MLC_2F and MLC_3F, and the masseter four, MLC_1S, MLC_2S, MLC_1F, and, in addition, an embryonic myosin light chain, MLC_temb, which is usually not present in normal adult skeletal muscle. These results probably reflect the way the muscles have evolved to meet the specialized functional requirements imposed upon them and are in agreement with the previously proposed concept that jaw and limb muscles belong to two distinct allotypes.     

Distinctive glycinergic currents with fast and slow kinetics in thalamus

We examined functional properties of inhibitory postsynaptic currents (IPSCs) evoked by medial lemniscal stimulation, spontaneous IPSCs (sIPSCs), and single-channel, extrasynaptic currents evoked by glycine receptor agonists or gamma-aminobutyric acid (GABA) in rat ventrobasal thalamus. We identified synaptic currents by reversal at E(Cl) and sensitivity to elimination by strychnine, GABA(A) antagonists, or combined application. Glycinergic IPSCs featured short (about 12 ms) and long (about 80 ms) decay time constants. These fast and slow IPSCs occurred separately with monoexponential decays, or together with biexponential decay kinetics. Glycinergic sIPSCs decayed monoexponentially with time constants, matching fast and slow IPSCs. These findings were consistent with synaptic responses generated by two populations of glycine receptors, localized under different nerve terminals. Glycine, taurine, or beta-alanine applied to excised membrane patches evoked short- and long-duration current bursts. Extrasynaptic burst durations resembled fast and slow IPSC time constants. The single, intermediate time constant (about 22 ms) of GABA(A)ergic IPSCs cotransmitted with glycinergic IPSCs approximated the burst duration of extrasynaptic GABA(A) channels. We noted differences between synaptic and extrasynaptic receptors. Endogenously activated glycine and GABA(A) receptor channels had higher Cl- permeability than that of their extrasynaptic counterparts. The beta-amino acids activated long-duration bursts at extrasynaptic glycine receptors, consistent with a role in detection of ambient taurine or beta-alanine. Heterogeneous kinetics and permeabilities implicate molecular and functional diversity in thalamic glycine receptors. Fast, intermediate, and slow inhibitory postsynaptic potential decays, mostly attributed to cotransmission by glycinergic and GABAergic pathways, allow for discriminative modulation and integration with voltage-dependent currents in ventrobasal neurons.