Experimental myotonia in mammalian skeletal muscle: Changes in contractile properties

Summary 2,4-Dichlorphenoxyacetate(2,4-D) produces symptoms very similar to those of clinical myotonia, not onlyin vivo but alsoin vitro. We studied contractions of strips from rat diaphragm that were made myotonic by addition of 2.5 mM 2,4-D to the bathing solution. Two effects of 2,4-D on the shape of the myogram are prominent: 1. The peak amplitude of single contractions increases by a factor of up to 6.2. The duration of relaxation is prolonged 10–20 fold. The myotonic symptom is more strongly elicited by stimulation with a brief pulse train rather than with a single pulse. A conditioning stimulus, given 0.5–4 s before the test stimulus, has contrary effects on the myotonic myogram: 1. The peak of contraction is further potentiated (post-tetanic potentation). 2. The prolonged relaxation is shortened to nearly normal duration (warm-up phenomenon). The results are discussed in relation to clinical observations.This work was supported by the Deutsche Forschungsgemeinschaft.     

A post-tetanic decrease of membrane resistance in mammalian skeletal muscle fibres and its antimyotonic effects

Summary The duration of the after-depolarization following an action potential becomes shorter when rat skeletal muscle is stimulated repetitively. This is particularly conspicuous when a muscle has been made myotonic, e.g. by addition of 1 mM 2,4-D to the bathing solution, because in myotonic muscle the duration of the afterdepolarization is longer than in normal muscle. The accelerated decline of afterdepolarization is associated with a temporary decrease of the specific membrane resistance (R _m ) following activity. The decrease ofR _m is more pronounced in myotonic than in nonmyotonic muscle. It becomes greater when activity is increased from 3 to 8 action potentials. In myotonic fibres which on the average have membrane resistances more than twice as large as normal,R _m can thus drop to normal values. The decrease of membrane resistance persists for more than a second after activity. We suggest that this temporary change ofR _m is the basis of the termination of a myotonic burst and for the warm-up phenomenon.This work was supported by the Deutsche Forschungsgemeinschaft.     

Training-induced changes in membrane transport proteins of human skeletal muscle

Training improves human physical performance by inducing structural and cardiovascular changes, metabolic changes, and changes in the density of membrane transport proteins. This review focuses on the training-induced changes in proteins involved in sarcolemmal membrane transport. It is concluded that the same type of training affects many transport proteins, suggesting that all transport proteins increase with training, and that both sprint and endurance training in humans increase the density of most membrane transport proteins. There seems to be an upper limit for these changes: intense training for 6–8 weeks substantially increases the density of membrane proteins, whereas years of training (as performed by athletes) have no further effect. Studies suggest that training-induced changes at the protein level are important functionally. The underlying factors responsible for these changes in transport proteins might include changes in substrate concentration, but the existence of “exercise factors” mediating these responses is more likely. Exercise factors might include Ca²⁺, mitogen-activated protein kinases, adenosine monophosphate kinases, other kinases, or interleukin-6. Although the magnitudes of training-induced changes have been investigated at the protein level, the underlying signal mechanisms have not been fully described.     

Fast to slow transition induced by experimental myotonia in rat EDL muscle

Experimental myotonia was induced by feeding rats with 20,25-diazacholesterol for up to 8 months. Histochemical analysis of myotonic extensor digitorum longus (EDL) muscle showed a progressive decrease of type IIB fibres and a concomitant increase of type IIA and type I fibres. A transient hypertrophy of type IIA fibres was observed 6 months after beginning the treatment. Analysis of the pattern of myosin light chains of single fibres from EDL showed that myotonia caused a progressive decrease of fibres showing a pure fast myosin light chain pattern and an increase of fibres showing coexistence of fast and slow myosin light chains (intermediate fibres). Only a small percentage of intermediate fibres showed coexistence of fast and slow myosin heavy chains. Myotonic fibres presented an increased sensitivity to caffeine which approached that of normal soleus fibres. Furthermore, sarcoplasmic reticulum (SR) vesicles isolated from hind limb fast muscles of myotonic rats demonstrated a decrease of Ca²⁺-dependent ATPase and Ca²⁺-transport activities as well as a decrease of immunoreactivity with anti-rabbit SR fast Ca²⁺-ATPase antibody. These results suggest that the increased electrical activity brought about by 20,25-diazacholesterol-induced myotonia, caused a fast to slow transition in the phenotypic expression of myosin and sarcoplasmic reticulum proteins.     

A special form of automaticity in the isolated papillary muscle of rats with experimental myotonia

Summary Myotonia of skeletal muscle was induced in rats by feeding 20, 25-diazacholesterol. All isolated papillary muscles of these rats showed spontaneously or following electrical stimulation a characteristic periodical automaticity of maximal 45 sec duration. The frequencypattern in these periods was the same as in the volleys of repetitive spiking in the myotonic skeletal muscle fibre. The so called warm up phenomenon could be demonstrated in the papillary muscles of myotonic rats as well. The results support the view, that in myotonia of skeletal muscle the membrane properties of the heart muscle are similarly changed.This work was presented in part at the Fifth Intern. Congress on Pharmacology, San Francisco, July 1972.     

The membrane capacity of mammalian skeletal muscle fibres

Summary Membrane capacity was measured as a function of fibre diameter in mammalian skeletal muscle fibres under normal conditions and under conditions designed to reduce the membrane chloride conductance, i.e. in solutions in which choride ions were replaced by sulphate or methylsulphate ions, or in normal Krebs solutions containing 2,4-dichlorophenoxyacetic acid (2.5mm). The experiments were done on rat sternomastoid, extensor digitorum longus and soleus muscle fibres. The average membrane capacity of fibres in each muscle was greater than normal when chloride conductance was reduced and the slope of the relationship between membrane capacity and fibre diameter increased. The results were consistent with the hypothesis that the space constant of the transverse tubule system in mammalian fibres is normally short because the transverse tubule membrane has a high chloride conductance. The experimental results imply that the space constant of the transverse tubule system was less than 40 µm for fibres in normal Krebs solution and greater than 100 µm for fibres with low membrane chloride conductance. The space constant was calculated using measured geometrical parameters of the transverse tubule, and measured membrane conductance, and the values were close to 20 µm for fibres in normal Krebs solution and between 50 and 120 µm for fibres with low chloride conductance.     

Alterations in calcium homeostasis reduce membrane excitability in amphibian skeletal muscle

The effects of alterations in intracellular calcium homeostasis on surface membrane excitability were investigated in resting Rana temporaria sartorius muscle. This was prompted by initial results from a fatiguing stimulation protocol study that demonstrated a fibre subpopulation in which action potential generation in response to a standard 1.5 V electrical stimulus failed despite mean membrane potentials [E _m, −69±2.3 mV (n=14)] compatible with spike firing in a control set of quiescent muscle fibres. Intracellular micro-electrode recordings showed a similar reversible loss of excitability, attributable to an increased threshold, despite only small (7.1±1.8 mV) positive changes in E _m after approximately 60-min exposures to nominally 0 Ca²⁺ Ringer solutions in which Ca²⁺ was replaced by Mg²⁺. This effect was not reproduced by addition of Mg²⁺ to the Ringer solution and persisted under conditions of Cl⁻ deprivation. The effects of three pharmacological agents, cyclopiazonic acid (CPA), caffeine and 4-chloro-m-cresol (4-CmC), each known to deplete store Ca²⁺ and increase cytosolic Ca²⁺ through contrasting mechanisms without influencing E _m, were then investigated. All three agents produced a more rapid, but nevertheless still reversible, loss of membrane excitability than in 0 Ca²⁺ Ringer solution alone. This reduction in membrane excitability persisted in fibres studied in solutions containing a normal [Ca²⁺] following prior depletion of store Ca²⁺ using CPA- and 4-CmC-containing solutions. These novel findings suggest that sarcoplasmic reticulum Ca²⁺ content profoundly influences surface membrane excitability, thereby providing a potential mechanism by which spike firing fails in well-polarised fibres during fatigue.The authors Usher-Smith and Xu were equal contributors to this paper.     

Developmental changes of membrane electrical properties in a rat skeletal muscle cell line.

1. The developmental changes of the cell membrane electrical properties were studied with micro-electrodes in a rat skeletal muscle cell line. 2. The resting potentials in myoblasts were minus 71 plus or minus 3 mV (mean plus or minus S.D.) and those in myotubes which were formed by fusion of myoblasts were minus 69 plus or minus 3 mV. There was no developmental change in the resting potential during the period examined. 3. The ionic mechanism for the resting potential was the same in myoblasts and myotubes. The resting membrane was almost exclusively permeable to K ions, while permeability to Na ions was not detectable. There was a small permeability to Cl ions. 4. The specific membrane resistance and capacitance were 8 k omega. cm-2 and 1 muF/cm-2 for myoblasts and 12 k omege. cm-2 and 5 muF/cm-2 for myotubes, respectively. 5. Action potentials in myoblasts were evoked by anode break stimulation. They were small and did not overshoot zero membrane potential. The action potentials in myotubes were larger, and had an average overshoot of 32 plus or minus 7 mV and a maximum rate of rise of 93 plus or minus 28 V/sec. 6. The current-voltage relation was examined. Delayed rectification was found in myotubes but not in myoblasts.     

Early membrane depolarization of the fast mammalian muscle after denervation

Summary The first alteration noted after denervation of the extensor digitorum longus muscles of rats was a decrease in resting membrane potential (RMP) which occurred at about 2h. The exact time course of this membrane depolarization was dependent upon the intramuscular length of the degenerating nerve stump. The decrease in RMP occurred prior to any detectable alteration in adenosine triphosphate and phosphocreatine content of the muscle. Prior to failure of spontaneous transmitter release, which occurred 10 h after denervation, some muscle fibres showed an increase in frequency of the miniature end-plate potentials (m.e.p.p.) with no alterations in amplitude and shape of the single potentials. Appearance of areas sensitive to acetylcholine (ACh) on the extra-junctional membrane occurred at 24 h after crushing the motor nerve. At 48 h after denervation a high ACh-sensitivity appeared at the muscle-tendon region, but was not detected in the majority of the muscle fibres studied midway between end-plate region and muscle-tendon area. The transverse resistance of a unit area of the muscle membrane was increased 3 days after denervation.These results provide evidence that the processes of post-denervation changes occur in the following temporal sequence: a) partial depolarization of the postsynaptic muscle membrane; b) a decrease and subsequent cessation of the spontaneous transmitter release preceded in some fibres by a transient increase in m.e.p.p. frequency; c) appearance of extrajunctional ACh sensitivity; d) increase in the transverse resistance of a unit area of the muscle membrane. It is suggested that the motor nerve releases more than one neurotrophic substance.Recipient of a Buswell Fellowship from the State University of New York at Buffalo and on leave of absence from the Christian-Albrechts-Universität, Kiel, West Germany.     

Silver ion-induced tension development and membrane depolarization in frog skeletal muscle fibres

Silver ions elicit dose-dependently a transient contracture in single fibres of bull-frog toe muscle placed in 0-Ca²⁺, Cl^–-free MOPS solution containing 3 mM Mg²⁺ and NO ₃ ^– . To elucidate the mechanisms involved, changes in membrane potential and in tension development were continuously measured following exposure to Ag⁺. The effect of Ag⁺ on contraction in fibres in which the membrane had been depolarized by elevating the external K⁺ concentration was also examined. The major findings of this investigation are as follows. (1) The mechanical threshold was shifted towards more negative potentials by 5 mV (–51 to –56 mV), when Ca²⁺ and Cl^– in the Ringer's solution were replaced with Mg²⁺ and NO ₃ ^– , respectively. (2) On the exposure of the fibres to 5 M Ag⁺, the membrane potential decreased by 1.6 mV from –87.8 mV and tension was developed. (3) In fibres soaked in a solution containing 10 mM K⁺ (corresponding to a membrane potential of –69.5 mV), 5 M Ag⁺ produced a large contracture similar to that seen in the control solution. (4) The Ag⁺-induced contracture was inactivated when more than 20 mM K⁺ was used. (5) The membrane depolarization evoked by either 20 or 50 M Hg²⁺ did not produce contraction. (6) Muscle fibres which had been exposed to 20 M Hg²⁺ for 5 min responded to 5 M Ag⁺ by a transient tension development. These findings strongly suggest that Ag⁺-induced tension development is not associated with depolarization of the surface membrane but rather is caused by specific actions of Ag⁺ on membrane proteins in the T-tubules.     

Immobilization atrophy and membrane properties in rat skeletal muscle fibres

Wet mass, resting membrane potential, frequency of miniature end-plate potentials and the concentration of [³H]ouabain-binding sites were studied after 7 days' immobilization of the rat soleus and extensor digitorum longus (EDL) muscles in the shortened or stretched position and after 3 and 7 days of remobilization. We observed that the loss of muscle mass by 37% in the rat soleus immobilized for 7 days in the shortened position is accompanied by a membrane depolarization of about 5 mV, a decrease in frequency of miniature end-plate potentials by 60 % and a decrease of [³H]ouabain binding by 25%. Only minor changes were found in stretched soleus and in shortened and stretched EDL. After 3 days of remobilization of stretched soleus the muscle mass, [³H]ouabain binding and miniature end-plate potential frequency recovered to control values but the resting membrane potential continued to decrease. All changes induced by immobilization disappeared on day 7 of remobilization.     

Changes in resting membrane potential and contractility of innervated and denervated skeletal muscle free grafts in the rat

Summary In free orthotopic auto-grafts of the extensor digitorum muscle of rats a marked temporary decrease of resting membrane potential (RMP) of two superficial layers of muscle fibres is observed at 2 days with subsequent recovery 4 days after transplantation. Such a temporary decrease of the RMP is not observed in grafts of denervated muscle. This difference in change of RMP is apparently related to a temporary marked decrease or loss of contractility observed in innervated but not denervated graft and may explain in part the relatively more successful grafting of denervated muscles.     

Calcium model for mammalian skeletal muscle

A model is presented describing quantitatively the events between excitation and force development in skeletal muscle. It consists of a calcium mediated activation model (c.m.a.m.) in series with a force generator model (f.g.m.). The c.m.a.m. was based on intracellular processes such as cisternal Ca-release, Ca-troponin interaction and Ca-uptake by SR. Ca-troponin complex concentration is an output of the c.m.a.m. and input of the f.g.m., the latter being a simplified actinmyosin cross-bridge model. Simulated and measured forces were compared for the rat slow (soleus) muscle. With the present structure and parameter values there is reasonable agreement between simulated and measured forces for single- and double-pulse responses. The parameters of the f.g.m. mainly determine the rising phase of the twitch since the Ca-release is brief and the binding of Ca to troponin is fast. In relaxation the Ca-troponin interaction and the Ca transporting ATPase parameters are also important. The behaviour of the muscle after a second action potential depends strongly on the level of Ca occupancy for both the troponin and Ca transporting ATPase.