Development of homogeneous fast and slow motor units in the neonatal mouse soleus muscle

We studied the fiber type composition and contractile properties of mouse soleus motor units at 2 days, 5 days and 2 weeks of age. We used Lucifer Yellow injection to mark muscle fibers belonging to the same motor unit in the two youngest age groups, and the traditional method of glycogen depletion in the oldest. The age groups were chosen because 2 days is at the end of muscle fiber production; 5 days is at the start of synapse elimination in the muscle and 2 weeks is at the end. Muscle fibers were classified as fast (F) or slow (S) on the basis of their myosin heavy chain (MHC) content, as determined by different monoclonal antibodies. Motor units are already dominated by either F- or S-fibers at 2 days, suggesting an early preferential innervation of the two types of fibers. A substantial part of the remaining refinement of the innervation takes place during the next 3 days, while the total number of terminals in the muscle remains constant. This is most easily explained by an exchange of aberrant for correct synapses during this period. A smaller part of the refinement of the innervation occurs during the subsequent period of synapse elimination.     

Length-dependence of isometric twitch tension potentiation and myosin phosphorylation in mouse skeletal muscle

The effect of changes in muscle length on post-tetanic isometric twitch tension potentiation and myosin P-light chain phosphorylation-was studied at 23°C in the mouse extensor digitorum longus muscle. The length-tension relationship was determined for the same muscles after a 30 min period of quiescence and between 30 s and 3 min after a 1.5 s tetanus at L₀. Isometric twitch tension is increased at all muscle lengths after the tetanus; however, the fractional increase in twitch tension rises from 0.2 at L₀ to a maximum of 0.3 at 1.2 L_0. The fractional increase in twitch tension measured at any fixed muscle length is constant between 30 s and 3 min post-tetanus. P-light chain phosphorylation remains constant between 30 s and 3 min post-tetanus followed by a slow decline to basal values. Under fixed length conditions, there is linear relationship between the relative magnitude of the twitch tension and the extent of P-light chain phosphor-ylation. Net myosin phosphorylalion measured after a 1.5 s tetanus at 1.23 L₀ is 35% less than that obtained under the same conditions at L_0. Thus, contraction-induced phosphorylation of P-light chain decreases with increased muscle length and post-tetanic potentiation at a constant level of P-light chain phosphorylation increases with increasing muscle length. These observations may be consistent with alterations in the sarcoplasmic Ca²⁺ ion transient as the muscle is lengthened.     

Simulation of post-tetanic potentiation and fatigue in muscle using a visco-elastic model

Post-tetanic potentiation (PTP) in single motor units was simulated using a simple visco-elastic model. Single isometric twitches and unfused tetani were obtained using a wide range of physiological input rates. Values of model parameters were chosen to simulate contraction times close to those of fast and slow muscle fibers. PTP has been attributed either to i) an augmented plateau level of active state or ii) an increase in time constant of active state decay. Our results show that a prolonged decay time of active state can account for most of the experimental data obtained in amphibian and mammalian preparations. In particular, potentiation is more marked in unfused tetani than in single twitches. Moreover the model accounts for PTP even in the case of a reduction of active state plateau due to fatigue.     

Distribution and structure-function relationship of myosin heavy chain isoforms in the adult mouse heart

The two cardiac myosin heavy chain isoforms, alpha and beta, exhibit distinct functional characteristics and therefore may be distributed regionally within the heart to match the functional demands of a specific region. In adult mouse hearts, which predominantly express alpha-myosin heavy chain, we observed high concentrations of beta-myosin in distinct areas such as at the tip of papillary muscles and at the base close to the valvular annulus. In light of these distinct distribution patterns of the myosin isoforms, we subsequently explored the isoform-specific structure-function relationships of the myosins. The alpha- and beta-isoforms are 93% identical in amino acid sequence, but it remains unclear which of the nonidentical residues determines isoform functionality. We hypothesized that residues situated within or close to the actin-binding interface of the myosin head influence actin binding and thereby modulate actin-activated ATPase activity. A chimeric myosin was created containing beta-sequence from amino acid 417 to 682 within the alpha-backbone. In mice, approximately 70% of the endogenous cardiac protein was replaced with the chimeric myosin. Myofibrils containing chimeric myosin exhibited ATPase activities that were depressed to the levels observed in hearts expressing approximately 70% beta-myosin. In vitro motility assays showed that the actin filament sliding velocity generated by chimeric myosin was similar to that of alpha-myosin, almost twice the velocities observed with beta-myosin. These data indicate that this large domain sequence switch conferred beta-like actin-activated ATPase activities to the chimeric myosin, suggesting that this region is responsible for the distinct hydrolytic properties of these myosin isoforms.     

Impairment of human soleus motor units during ischemia

It is generally accepted that ischemia produced by limb compression affects rapidly conducting large-diameter Ia afferents in the early stage and that the motor nerve-muscle complex is blocked later. This notion, however, seems to be controversial for several reasons, so an attempt to reveal the amount of motor unit (MU) impairment during ischemia was made. Observation of human soleus muscle electromyographic (EMG) signal recorded either by bipolar needle electrode or by surface electrodes at various levels of voluntary contraction during the course of ischemia showed that low-threshold small MUs were affected first while high-threshold large MUs survived longer. The changes in EMG patterns were temporally correlated with T-reflex deterioration. It is suggested that the early loss of low-threshold MUs may play a definite role in alterations of reflexes during ischemia.     

Twitch potentiation and caffeine contractures in isolated rat soleus muscle

1. Electrically-evoked twitch and tetanic tension were measured in isolated rat soleus muscle after exposure to caffeine. 2. Between 0.01 and 2.5 mM caffeine twitch tension was potentiated, reaching a peak of 150% of Resting Tension at 0.5 mM. 3. Biphasic Tension development with relaxation was observed at 2.5 mM caffeine with maximal contractures (110% tetanic tension) occurring at 20 mM. 4. Creatine phosphate and ATP stores were maintained throughout the period of tension development and relaxation. 5. In contrast with amphibian muscle, the isolated soleus is very sensitive to low doses of caffeine and produces biphasic caffeine contractures which relax in the presence of caffeine.     

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

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

Molecular mechanics of mouse cardiac myosin isoforms

Two myosin isoforms are expressed in myocardium, alphaalpha-homodimers (V(1)) and betabeta-homodimers (V(3)). V(1) exhibits higher velocities and myofibrillar ATPase activities compared with V(3). We also observed this for cardiac myosin from normal (V(1)) and propylthiouracil-treated (V(3)) mice. Actin velocity in a motility assay (V(actin)) over V(1) myosin was twice that of V(3) as was the myofibrillar ATPase. Myosin's average force (F(avg)) was similar for V(1) and V(3). Comparing V(actin) and F(avg) across species for both V(1) and V(3), our laboratory showed previously (VanBuren P, Harris DE, Alpert NR, and Warshaw DM. Circ Res 77: 439-444, 1995) that mouse V(1) has greater V(actin) and F(avg) compared with rabbit V(1). Mouse V(3) V(actin) was twice that of rabbit V(actin). To understand myosin's molecular structure and function, we compared alpha- and beta-cardiac myosin sequences from rodents and rabbits. The rabbit alpha- and beta-cardiac myosin differed by eight and four amino acids, respectively, compared with rodents. These residues are localized to both the motor domain and the rod. These differences in sequence and mechanical performance may be an evolutionary attempt to match a myosin's mechanical behavior to the heart's power requirements.     

Pig brain homogenates contain smooth muscle myosin and cytoplasmic myosin isoforms

Summary Three myosin isoforms, two of smooth muscle and one of cytoplasmic origin, were found in porcine brain by Western blotting analysis with antibodies specific for smooth and cytoplasmic myosins. The smooth muscle isoforms comprise at least 30% of the total myosin present. Brain tissue is therefore not a suitable source for the isolation of pure cytoplasmic contractile proteins.Abbreviations DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - FITC fluorescein isothiocyanate - PBS phosphate buffered saline - SDS PAGE polyacrylamide electrophoresis in the presence of sodium dodecylsulphate - TRIS tris(hydroxymethyl)aminomethane     

Populations of motor units of the human soleus muscle supplying H reflex generation: Electrophysiological analysis

Variations in the amplitude of H and M responses of them. soleus related to the variation in intensity of stimulation of then. tibialis comm. were evaluated in five persons with different ratios of the maximum H and maximum M response amplitudes (from 0.27 to 0.75). A decrease in amplitude of the H reflex accompanied by an increase of M response is supposed to be determined by collision of ortho- and antidromically conducted spikes in motoneuronal axons; this makes it possible to quantify the participation of various motoneuronal populations differing in activation thresholds of their axons in H reflex generation. The H response in individuals with a low ratio of the maximum H and M response amplitudes was shown to be due primarily to the involvement of high-threshold motoneurons. When the ratio between the above-mentioned maximum EMG potentials was high, all populations of motoneurons, except very low-threshold ones, participated in the H reflex generation. In all cases, only a portion of high-threshold motoneurons was involved in H activity, which contradicts the so-called size principle.Neirofiziologiya/Neurophysiology, Vol. 25, No. 6, pp. 417–420, November–December, 1993.     

Influence of neonatal motor denervation on expression of myosin heavy chain isoforms in rat muscle spindles

In order to evaluate the effects of fusimotor elimination on the expression of myosin heavy chain (MHC) proteins in intrafusal fibres, we compared the muscle spindles in hind limb muscles of 3- to 6-week-old rats de-efferented at birth with those of their litter-mate controls. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal MHC isoforms, against synaptophysin, the neurofilament 68 kD subunit and laminin. We found that de-efferented intrafusal fibres differentiated, as in normal spindles, into nuclear bag and bag fibres both containing predominantly slow MHC, and nuclear chain fibres that contained fast and neonatal MHC. In both de-efferented and control intrafusal fibres the same MHCs were stained; the degree and extent of staining, however, varied. Both types of de-efferented bag fibres displayed a high content of slow tonic and slow twitch MHC along most of the fibre length, in contrast to the prominent regional variation in control bag fibres. In their encapsulated regions, the de-efferented bag fibres were more similar to each other in their reactivity to anti-fast twitch and anti-neonatal MHC antibodies than the control bag fibres. In these aspects they resembled more closely the bag fibres of newborn rats. The differences might be due to an arrest of "specialization" in the regional expression of the different MHC isoforms. Chain fibres developed MHC patterns identical to those of control spindles with all the antibodies used, even though they differentiated from the beginning in the absence of motor innervation. The structural differentiation of the capsule and sensory innervation in de-efferented muscle spindles, as shown by anti-laminin, anti-synaptophysin and anti-neurofilament staining, did not differ from the controls. We conclude, in agreement with previous studies, that the sensory innervation plays a key role in inducing and supporting the differentiation of intrafusal fibres and the specific expression of their MHC. However, we also show that motor innervation and/or muscle function seem to be necessary for the diversity in the expression and distribution of different slow and fast MHC isoforms in the bag and bag fibres.     

Influence of neonatal motor denervation on expression of myosin heavy chain isoforms in rat muscle spindles

Summary In order to evaluate the effects of fusimotor elimination on the expression of myosin heavy chain (MHC) proteins in intrafusal fibres, we compared the muscle spindles in hind limb muscles of 3- to 6-week-old rats de-efferented at birth with those of their litter-mate controls. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal MHC isoforms, against synaptophysin, the neurofilament 68 kD subunit and laminin. We found that de-efferented intrafusal fibres differentiated, as in normal spindles, into nuclear bag₁ and bag₂ fibres both containing predominantly slow MHC, and nuclear chain fibres that contained fast and neonatal MHC. In both de-efferented and control intrafusal fibres the same MHCs were stained; the degree and extent of staining, however, varied. Both types of de-efferented bag fibres displayed a high content of slow tonic and slow twitch MHC along most of the fibre length, in contrast to the prominent regional variation in control bag fibres. In their encapsulated regions, the de-efferented bag fibres were more similar to each other in their reactivity to anti-fast twitch and anti-neonatal MHC antibodies than the control bag fibres. In these aspects they resembled more closely the bag fibres of newborn rats. The differences might be due to an arrest of specialization in the regional expression of the different MHC isoforms. Chain fibres developed MHC patterns identical to those of control spindles with all the antibodies used, even though they differentiated from the beginning in the absence of motor innervation.The structural differentiation of the capsule and sensory innervation in de-efferented muscle spindles, as shown by anti-laminin, anti-synaptophysin and anti-neurofilament staining, did not differ from the controls.We conclude, in agreement with previous studies, that the sensory innervation plays a key role in inducing and supporting the differentiation of intrafusal fibres and the specific expression of their MHC. However, we also show that motor innervation and/or muscle function seem to be necessary for the diversity in the expression and distribution of different slow and fast MHC isoforms in the bag₁ and bag₂ fibres.     

Relationship between myoglobin and succinate dehydrogenase in mouse soleus and plantaris muscle fibres

Summary This report describes a quantitative histochemical study of myoglobin in skeletal muscle fibres. The muscle fibres were classified as fast or slow on the basis of their quantitative myofibrillar ATPase histochemistry. A large range of myoglobin absorbance values was found among fast skeletal muscle fibres. This range was relatively small among slow fibres. The concentrations of myoglobin and the activities of succinate dehydrogenase in individual muscle fibres in serial sections are weakly correlated in both the mouse soleus and plantaris muscle. The myoglobin concentration is higher in fast and slow oxidative soleus muscle fibres and the succinate dehydrogenase activity in these fibres is lower than in oxidative plantaris muscle fibres in the same range of cross-sectional area.     

The myosin motor: muscle contraction and in vitro movement

The molecular mechanism of in vitro movement is assumed, by most investigators, to be identical to that of muscle contraction. We discuss this view, which raises various problems. We believe there are mechanisms for muscle contraction (in this case considerable forces are developed, with small displacements) and other mechanisms for in vitro movement (giving large displacements, without necessarily generating substantial forces). Hybrid models may explain muscle contraction. The traditional swinging-crossbridge model may explain in vitro movement. For muscle contraction, movement may result partly from the swinging-crossbridge mechanism and partly from other factors. Comparisons of different fibres at different moments of the Mg-ATPase cycle suggest that both the value of the isometric force in muscle and in vitro and that of the Mg-ATPase activity used in vitro need to be reconsidered. The recently reported dependence of the isometric active tension of smooth skinned fibres on temperature appears to be weaker than predicted by the swinging-crossbridge theory alone. This recent observation is compatible with the existence of other forces (electrostatic repulsions) decreasing with temperature as has been known for some years. From recent experimental data, we think the biochemistry of myosin and actomyosin should be reassessed, to try to find new details of the mechanisms of muscle contraction and in vitro motility.     

Early and late post-tetanic potentiation,and post-tetanic block in a monosynaptic reflex pathway

Observations have been made upon the nature of early and late post-tetanic potentiation and upon post-tetanic block of presynaptic collaterals with particular reference to behavior in circumstances of varied duration and frequency of conditioning stimulation. Early potentiation is most conspicuous following brief tetani at high frequency, late potentiation following long tetani, much lower frequencies being all that are needed. The two phenomena thus are distinguishable and separate. Dorsal root electrotonus produced by stimulations of varying duration and frequency is described, and the similarity between behavior of the D.R.IV R. electronic potential and early potentiation demonstrated. It is shown how early potentiation and post-tetanic block are due to the same process (hyperpolarization) at different intensities. The view that the agency for potentiation is associated with augmented presynaptic action due to hyperpolarization is confirmed. A diagram is constructed to indicate the probable temporal courses of early and late potentiation.