Reversible depression of action potentials and force production in frog single muscle fibres by calmodulin-inhibitors

The effects of the calmodulin-inhibitors trifluoperazine, thioridazine and zaldaride maleate on the responses to electrical stimulation in isolated frog skeletal muscle fibres were investigated. All three drugs initially reduced the amplitude of the action potentials but potentiated twitch force. This was followed by a total loss of action potentials and force production. However, the resting membrane potential was not changed. The effects were completely reversible upon removal of the drugs. These results suggest that an intact calmodulin system is required for normal function of the sarcolemmal sodium channels of frog skeletal muscle.     

Phosphocreatine as a marker of contractile activity in single muscle fibres

ATP and phosphocreatine (PCr) were measured in randomly selected single fibres from control, 1- and 8-day low-frequency-stimulated rabbit tibialis anterior muscles. The fibres were classified according to their myosin heavy chain (MHC) complement as type I, IIA or IID. In 1-day stimulated muscle, which has previously been shown to exhibit a steep decline in force output, two fibre populations could be distinguished according to either normal or markedly depressed PCr levels. The fibre population exhibiting normal PCr levels encompassed a major fraction (65%) of type IID fibres and a minor fraction (35%) of IIA fibres. The population with reduced PCr levels comprised type I fibres (@50% reduced), the majority of type IIA fibres (@80% reduced), and a minor fraction of type IID fibres (@70% reduced). Levels of ATP were unaltered in type I and IIA fibres, but were @ 20% reduced in those IID fibres that exhibited low PCr levels. Assuming that those fibres that displayed reduced PCr levels were contracting, the IID and IIA fibres with normal PCr levels were regarded as metabolically recovering, non-contracting fibres. As previously shown, these fibres are transiently refractory during the early phase of low-frequency stimulation. After 8 days of chronic low-frequency stimulation, when force was shown to rise again, most fibres appeared more uniform with regard to reduced PCr and ATP levels. Our results suggest that PCr can be used as a sensitive measure of the degree of activity in single-fibre studies. Received: 14 January 1999 / Received after revision and accepted 21 April 1999     

The mechanism of oscillatory activity at low membrane potentials in cardiac Purkinje fibres

1. The mechanism of oscillations at low membrane potentials in Purkinje fibres has been investigated using voltage clamp experiments.2. The oscillations are generated by time-dependent variations in an outward current component, i(x1), that is activated over the voltage range -40 to 10 mV. During normal activity, this current is responsible for initiating full repolarization to the resting potential (Noble & Tsien, 1969b) so that the oscillations represent a failure of the normal repolarization process, probably as a consequence of a small change in background (leakage) current.3. These oscillations are distinct from the normal pacemaker activity of Purkinje fibres which is generated by a separate time-dependent current, i(K2) (Noble & Tsien, 1968). i(K2) shows no time-dependence when the membrane potential variations are entirely positive to -65 mV and cannot, therefore, be involved in the oscillatory activity apart from contributing a background outward current.4. The amplitude and frequency of the oscillations are very sensitive to applied currents less than 1 muA/cm(2). Larger currents abolish the oscillatory activity.5. The mechanism of the oscillations is discussed in relation to the possible mechanisms underlying the natural pacemaker activity of the sino-atrial (SA) node.     

The maintenance of resting potentials in glycerol-treated muscle fibres

1. A modification of a previously published method for the disruption of the T-tubules of frog skeletal muscle is described. The modification permits the disruption of the T-tubules without the decline in resting potentials which was reported previously.2. The method for the disruption of the T-tubules involves the washout of glycerol following loading in a 400 mM glycerol Ringer solution. The modification consists of elevating the concentration of divalent cations in the Ringer used for glycerol washout.3. The optimum concentrations are 5 mM-Ca(2+) and 5 mM-Mg(2+) added as their chloride salts. Neither 10 mM-Ca(2+) nor 10 mM-Mg(2+) are as effective as the combination of each at 5 mM. Other concentrations gave less satisfactory results.4. The use of the modified technique provides a preparation which maintains 85-90 mV resting potentials for up to 6 or 8 hr but which will not contract in response to membrane depolarization.     

Calcium action potentials in innervated and denervated rat muscle fibres

We studied the generation of calcium action potentials (Ca APs) in innervated and denervated fibres of the extensor digitorum longus of the rat in a tetraethylammonium (TEA) sulphate solution plus 3–4 diaminopiridine (3–4 DAP). The main results are the following: (1) more than 90% of the innervated fibres were capable of developing well-sustained Ca APs that were blocked by Cd or nifedipine; (2) the incidence of Ca APs obtained from the denervated fibres was substantially lower than in the control preparations; (3) no relation was found between the appearance of Ca APs in the denervated fibres and the resting membrane potential (V _m), stimulus duration (500–2000 ms) or holding potential (–80, –100 mV); (4) The percentage of denervated fibres that exhibited Ca APs was increased significantly with the following procedures. First, by raising the external Ca concentration to 14 mM; second, by depleting the intracellular K concentration by overnight exposure of the muscles to a free K-Cs solution; (c) and third, by incubating the muscles in 500 nM apamin, a venom that inhibits the K conductance activated by Ca. Several factors may be involved in the lower incidence of Ca APs obtained in denervated fibres: (1) a diminished Ca current due to a reduction in the driving force as a result of an increment in the intracellular Ca concentration; (2) a persistence of a shunting K conductance that is not inhibited by TEA and 3–4 DAP; (3) a shift in the voltage dependence of the activation and inactivation parameters of the Ca current or the appearance of a new type of Ca channel with a different kinetics.     

Action potentials reconstructed in normal and myotonic muscle fibres.

1. Muscle fibres from goats with myotonia congenita show characteristic responses to stimulation with intracellular currents (Adrian & Bryant, 1974). To test whether the reduced surface chloride conductance can account for these myotonic discharges, we have calculated responses of a model 'muscle fibre' to intracellular current of long duration (greater than 100 msec), assuming that the current is applied at the end of the fibre, that the fibre is of finite length, that a regenerative action potential occurs in the transverse tubular system as well as the surface, and that the potassium current in the wall of the transverse tubular system raises the potassium in the tubular lumen. In the absence of information about the kinetic parameters of the ionic currents in mammalian muscle we have used numerical values from frog muscle (Adrian, Chandler & Hodgkin, 1970). 2. In calculations with a normal surface chloride conductance a long maintained current gives only one action potential. Reduction of the chloride conductance to a half produces repetitive firing during the current; reduction to a tenth produces repetitive firing during and a small number of action potentials after the end of the current. Elimination of the tubular potassium accumulation from the calculation reduces the number but does not eliminate action potentials arising after the end of the applied current. 3. With a tenth of the normal chloride conductance calculated responses show maintained firing following a constant current if the deactivating rate of the sodium channels (betam) is reduced by 25%. As before, eliminating potassium accumulation reduces the number of post-stimulus action potentials, but it does not eliminate them altogether. 4. We conclude that in the absence of a surface chloride conductance tubular potassium accumulation could certainly contribute to the instability of the membrane, but it is clear that potassium accumulation is not the only reason for the instability of myotonic muscle fibres. The kinetics of the sodium channels are important and we do not know that they are the same in normal and myotonic fibres. Nevertheless the presence of a surface chloride conductance does stabilize the response of a fibre to constant current or to repetitive stimulation, and its absence could be a sufficient condition for myotonic behaviour.     

Effects of diethyl-stilboestrol on single fibres of frog skeletal muscle

The effects of diethyl-stilboestrol (DES), one of the most potent estrogens, were studied on single muscle fibres of the frog. In relatively low concentrations (5 μM), DES greatly potentiates the twitch response of the fibre without significantly affecting the amplitude of the tetanus response. The twitch potentiation is accompanied by an increase in time to peak tension and a marked prolongation of the relaxation phase. In tetanic response the half decay time after the last stimulus is also prolonged by DES. DES causes no changes in the resting or action potential of the muscle fibre. The S-shaped curve relating peak contracture tension and caffeine concentration is shifted towards lower caffeine concentrations by DES. Dantrolene greatly suppresses the DES potentiated twitch. It is concluded that DES potentiates the twitch response and prolongs the relaxation time by inhibition of the calcium re-uptake by the sarcoplasmic reticulum.     

Simulation of single muscle fibre action potentials

Using the volume conductor model, a single muscle fibre action potential can be expressed as a convolution of the transmembrane current and a weighting function. By simplifying the weighting function, the line source model is derived. We have developed similar expressions to compute the single muscle fibre action potential using simple models and physical considerations without any mathematical complexity. The relationship between the conduction velocity and amplitude is analysed and it is concluded that, for a given fibre, the amplitude is inversely proportional to the conduction velocity. This agrees with the experimental data reported in the literature. The relation between amplitude and fibre diameter is studied. The amplitude increases with diameter owing to the increase in membrane current, but it is counteracted by the increase in conduction velocity. Because of the opposing effects, a point of inflection in the amplitude/diameter relationship is observed. Squareroot, square and linear dependencies of conduction velocity on fibre diameter were used. The difference in the peak-to-peak amplitude with these relationships is small and a linear relation between amplitude and fibre diameter seems reasonable irrespective of the conduction velocity/fibre diameter relationship. The effect of lumping the transmembrane current at the axis of the fibre is discussed. This approximation results in an underestimation of the peak-to-peak amplitude near the surface, and the error is close to 50% for very large fibres. This error is accounted for in the model. The recording distance at which the peak-to-peak amplitude of the signal is 100 μV is found to be 400 μm in the model. This is in good agreement with values obtained from a single-fibre electrode recording. The model is computationally fast and precise. It can easily be used with few modifications to simulate single fibre action potentials recorded using different electrodes.     

Miniature potentials in denervated slow muscle fibres of the frog

1. Pyriformis and iliofibularis muscles of the frog were studied with micro-electrodes. Both muscles contain a mixture of fast and slow muscle fibres, which can be distinguished by differences in their miniature end-plate potentials (min.e.p.p.s).2. In addition, the membrane time constant of fast fibres is < 20 msec, while that of slow fibres is > 200 msec.3. The characteristic difference in time constant persisted after denervation and allowed the identification of fibre type.4. Denervated slow fibres had min.e.p.p.s of variable time courses and skew amplitude distributions. Their frequency was lower than in normally innervated fibres, and could be increased by hypotonic solutions.5. In analogy with similar observations made previously in fast muscle fibres, it is suggested that after nerve degeneration the Schwann cells of small motor nerve fibres release packages of transmitter which give rise to min.e.p.p.s.     

Potentiometric measurement of membrane action potentials in frog muscle fibres

1. A potentiometric method for recording membrane action potentials from small segments of isolated skeletal muscle fibres has been described.2. The value of the method in muscle physiology has been appraised by recording from fast and slow skeletal muscle fibres of the frog under a variety of conditions.3. Artifacts associated with this method have been described and their sources investigated.     

Effects of diethyl-stilboestrol on single fibres of frog skeletal muscle.

The effects of diethyl-stilboestrol (DES), one of the most potent estrogens, were studied on single muscle fibres of the frog. In relatively low concentrations (5 muM), DES greatly potentiates the twitch response of the fibre without significantly affecting the amplitude of the tetanus response. The twitch potentiation is accompanied by an increase in time to peak tension and marked prolongation of the relaxation phase. In tetanic response the half decay time after the last stimulus is also prolonged by DES. DES causes no changes in the resting or action potential of the muscle fibre. The S-shaped curve relating peak contracture tension and caffeine concentration is shifted towards lower caffeine concentrations by DES. Dantrolene greatly suppresses the DES potentiated twitch. It is concluded that DES potentiates the twitch response and prolongs the relaxation time by inhibition of the calcium re-uptake by the sarcoplasmic reticulum.