Investigations of the Electrical Properties of Cardiac Muscle Fibres with the Aid of Intracellular Double-Barrelled Electrodes

Current has been passed through the cell membrane of muscle fibres of the isolated rabbit right ventricle with the aid of intracellular double-barrelled microelectrodes. Two types of muscle fibres were distinguished which are called P and V fibres. The relation between the intensity of a hyperpolarising current applied during the rising phase and the maximum amplitude of the action potential was different in these fibres. For P fibres the relation was essentially linear over most of the range of currents used. For V fibres the change in maximum action potential amplitude was either negligible or did not appear until a certain value of hyperpolarising current was reached. This behaviour of V fibres can be understood if a drop in polarisation resistance occurs during the rising phase and is of such short duration that the polarisation resistance has returned to its resting value before the crest of the action potential is reached. P fibres have an estimated mean resting polarisation resistance of (106 ± 13) K ohms, and a rheobase current strength of (0.08 ± 0.02) µa. In V fibres the resting polarisation resistance was (47 ± 29) K ohms and the rheobase current strength (0.47 ± 0.28) µa.     

Effect of glycerin removal on the electrical resistance of isolated muscle fiber in the frog

Fibres isolated from iliofibularis muscles of the frog Rana esculenta were studied under current-clamp conditions with a double sucrose-gap technique. An increase of membrane resistance in muscle fibres (Rm) was demonstrated during the first 10-15 min of glycerol removal in K2SO4 solution. After a 30 min treatment in glycerol-containing K2SO4 Rm was 1.40 +/- 0.12 k omega X X cm2. The transfer of muscles from the glycerol-K2SO4 solution to an isotonic K2SO4 solution resulted in a progressive increase in the Rm which after a 15 minutes removal of glycerol reached the mean value of 2.02 +/- 0.22 k omega X cm2. It is suggested that this increase may reflect detubulation of the fibres caused by glycerol removal in muscles.     

The effect of tetraethylammonium and the decrease in the extracellular chloride concentration on membrane depolarization and contraction of skeletal muscle fiber induced by a hyperpotassium solution

1.--The tetraethylammonium (TEA) effects on K+ contracture and membrane depolarization are compared in both crab and frog skeletal muscle fibres. 2.--The mechanical tension of the contracture is reduced by the TEA in frog skeletal muscle fibre; it is increased in crab skeletal fibre. 3.--When no mechanical phenomenon is observed in frog skeletal muscle, the amplitude and the velocity of membrane depolarization induced by an increase of outward K+ concentration is reduced by the TEA. These effects are in opposition in crab muscle fibre. 4.--In crab muscle fibre, the results obtained tend to show that the C1-ions are not distributed on each side of the membrane according to Donnan equilibrium.     

Effects of partial denervation on the distribution of acetylcholine receptors and other membrane properties in innervated and paralyzed fibers of rat skeletal muscle

The origin of the membrane changes induced in skeletal muscle by denervation has been investigated by examining partially denervated rat hindlimb muscles rendered inactive for 2-3 days by a chronic conduction block in the sciatic nerve. Extra-junctional sensitivity to acetylcholine and spike resistance to tetrodotoxin developed to the same extent in the denervated and the adjacent innervated but inactive fibres. On the other hand, impulse-blocked fibres of control muscles not containing denervated fibres showed, at this early time, little membrane changes. These results are interpreted as indicating that the response of muscle to denervation is due to the combined action of inactivity and products of nerve degeneration.     

The effect of Rb+, Cs+, and T1+ on the gramicidin A-induced conductance changes of the skeletal muscle cell membrane

The channel-forming antibiotic gramicidin A increases the K+ conductance of frog skeletal muscle fibres in isotonic K2SO4 solution. The conductance of the gramicidin channel is not affected by Rb+ or Cs+, but is reduced by T1+. In contrast, the conductance of the normal K+ channel is decreased by Rb+ and Cs+ but is nearly unaffected by 5 and 10 mM T1+. The results suggest differences in the cation permeation through the gramicidin and the K+ channel of the muscle cell membrane.     

Electrophysiological characteristics of muscle fibers during denervation

Experimental material obtained on mouse extensor digitorum longus was presented concerning the dynamics of changes in the membrane potential (MP) of muscle fibres after chronic denervation (1-12 days), the effect of ouabain and increase of extracellular potassium on MP of normal and denervated muscles, changes of input resistance and volumes of muscle fibres after denervation.     

Degeneration of the afferent fibers simulating the effects of motor denervation on skeletal muscle

Degeneration of afferent nerve fibres was induced in rats in order to observe its effects on the properties of the extra-junctional membrane of soleus muscle fibres. In one approach, removal of dorsal root ganglia L4 and L5 was accomplished in preparations with intact or impulse-blocked (with tetrodotoxin containing cuffs around the sciatic nerve) efferent innervation. Spike resistance to tetrodotoxin developed in the inactive deafferented preparations earlier and to a greater extent than in control, that is only impulse-blocked, preparations. In another series of experiments, efferent denervation alone proved to be less effective than the association of efferent and afferent denervation. On the other hand, section of the afferent fibres central to the dorsal root ganglia was without effect. These results are consistent with the interpretation that products of nerve degeneration contribute together with inactivity to the development of the extrajunctional membrane changes observed in skeletal muscle after denervation.     

Ionic and molecular mechanisms of beta-amyloid-induced depolarization of the mouse skeletal muscle fibres

Excess production and accumulation of beta-amyloid peptide (betaAP) are central for pathogenesis of Alzheimer's disease. Numerous studies showed that betaAP possessed wide range of toxic effects on neurons, however the mechanism of betaAP influence on another types of excitable cells, for example, skeletal muscle fibres, is unknown. In electrophysiological experiments on the mouse diaphragm, we found for the first time that betaAP (25-35 fragment, 10-6 M) disturbs the processes of the resting membrane potential generation in muscle fibres, leading to depolarization by two mechanisms: 1) inhibition of Na+,K(+)-ATPase, which leads to loss of impact of this pump to the resting membrane potential; 2) increase of membrane cationic permeability due to formation of "amyloid" channels blocked with Zn2+ ions. Our results significantly broaden current understanding of mechanisms of motor disturbances and skeletal muscle pathology in Alzheimer's disease, inclusion body myositis and other betaAP-related disorders.     

Ba2+ ions block K+-induced contractures by antagonizing K+-induced membrane depolarization in frog skeletal muscle fibres

The effects of Ba2+ ions on twitches, K+-induced contractures, and on intracellularly recorded membrane potentials (Em) and depolarizations of frog skeletal muscle fibres were investigated. Exposure of toe muscles to choline--Ringer's solution with 10(-3) M Ba2+ with Ca2+ (1.08 mM) eliminated or very greatly reduced contractures produced by 60 mM K+. In contrast, not only did the same concentration of Ba2+ ions fail to depress the twitch tension of isolated semitendinosus fibres when added to Ringer's with Ca2+, but it even restored twitches that had been eliminated in a zero Ca2+ Ringer's solution. The resting Em of sartorius muscle fibres in choline--Ringer's solution was reduced about 20 mV by 10(-3) M Ba2+. This Ba2+ ion concentration also antagonized the K+-induced depolarization. Thus in the presence of 1 mM Ba2+, 20 mM K+ hyperpolarized rather than depolarized the fibres and 60 or 123 mM K+ produced only very slowly developing, small depolarizations. These results suggest that the loss of the K+-induced contracture in choline-Ringer's caused by Ba2+ ions is due to an inhibition of the K+-induced depolarization. The latter result is consistent with previous findings of other workers that Ba2+ ions block membrane K+ channels.     

Expression of the skeletal muscle dystrophin-dystroglycan complex and syntrophin-nitric oxide synthase complex is severely affected in the type 2 diabetic Goto-Kakizaki rat

The inability of insulin to stimulate glucose metabolism in skeletal muscle fibres is a classic characteristic of type 2 diabetes. Using the non-obese Goto-Kakizaki rat as an established animal model of this type of diabetes, sucrose gradient centrifugation studies were performed and confirmed the abnormal subcellular location of the glucose transporter GLUT4. In addition, this analysis revealed an unexpected drastic reduction in the surface membrane marker beta-dystroglycan, a dystrophin-associated glycoprotein. Based on this finding, a comprehensive immunoblotting survey was conducted which showed a dramatic decrease in the Dp427 isoform of dystrophin and the alpha/beta-dystroglycan subcomplex, but not in laminin, sarcoglycans, dystrobrevin, and excitation-contraction-relaxation cycle elements. Thus, the backbone of the trans-sarcolemmal linkage between the extracellular matrix and the actin membrane cytoskeleton might be structurally impaired in diabetic fibres. Immunohistochemical studies revealed that the reduction in the dystrophin-dystroglycan complex does not induce obvious signs of muscle pathology, and is neither universal in all fibres, nor fibre-type specific. Most importantly, the expression of alpha-syntrophin and the syntrophin-associated neuronal isoform of nitric oxide synthase, nNOS, was demonstrated to be severely reduced in diabetic fibres. The loss of the dystrophin-dystroglycan complex and the syntrophin-nNOS complex in selected fibres suggests a weakening of the sarcolemma, abnormal signalling and probably a decreased cytoprotective mechanism in diabetes. Impaired anchoring of the cortical actin cytoskeleton via dystrophin might interfere with the proper recruitment of the glucose transporter to the surface membrane, following stimulation by insulin or muscle contraction. This may, at least partially, be responsible for the insulin resistance in diabetic skeletal muscles.     

The properties of the extraocular muscles of the frog. III. Morphological, mechanical and pharmacological properties of the isolated retractor bulbi muscle.

Some morphological, physiological, and pharmacological properties of the retractor bulbi muscle of the frog were tested. The enzyme-histochemical investigation shows that the retractor bulbi muscle contains twitch muscle fibres only. Two types of twitch muscle fibres, which are especially different in their diameter and in the content of mitochondria, build the muscle in an irregular arrangement; tonic muscle fibres were not observed. On the average, the isolated retractor bulbi muscle has at room temperature a contraction time of 26 ms, a half-relaxation time of 28 ms, a fusion frequency of 75 stimuli/s, and a twitch-tetanus ratio of 0.28. The fatigability of this muscle is higher than in oculorotatory eye muscles but lower than in skeletal muscles of the frog. An increase of the extracellular K+-concentration elicits in retractor bulbi muscles a quickly transient contracture; the mechanical threshold of the muscle fibres is found in a range between 20 and 25 mM K+ in Ringer solution. Similar short-lasting contractures, which are probably caused by twitch fibres, rich in mitochondria, are also evoked by application of depolarizing drugs like acetylcholine. The properties of the retractor bulbi muscle are compared with those of the sartorius muscle of the frog, which likewise contains twitch muscle fibres only.     

Activation of ATP-dependent K+ currents in intact skeletal muscle fibres by reduced intracellular pH.

We have used three-microelectrode voltage clamp in conjunction with the ammonium prepulse method to investigate the effects of lowered intracellular pH (pHi) on resting potassium currents of frog skeletal muscle fibres. Potassium currents were recorded in 40 mM K+, Cl(-)-free solution in response either to voltage steps or ramps. An ammonium prepulse (2 h) reduced pHi to 6.45 from a control value of 7.19. The intracellular ATP concentration, measured with high-pressure liquid chromatography (HPLC), was unchanged by this procedure. Mean outward potassium currents were larger in low pHi than in control fibres, being about twice as large at +40 mV, whereas mean inward currents were very similar in control and low-pHi fibres. The sulphonylurea glibenclamide blocked single KATP channels in excised patches with a Kd of 3 microM. In intact fibres 50 microM glibenclamide had no effect on K+ currents in controls but reduced currents in low-pHi fibres. In the presence of glibenclamide, K+ currents in low-pHi fibres were not significantly different from those in control fibres. We suggest that reduced pHi in intact skeletal muscle fibres opens ATP-dependent potassium channels (KATP channels), as has been shown to occur in excised patches of membrane.     

Analysis of changes in the membrane potential of denervated muscle

Quantitative analysis of the experimental data presented in the previous paper has shown that the electrogenic pump component of the membrane potential of muscle fibres on the third day after denervation is in the average 8.7 mV, and the diffusion component--12.9 mV lower than that in the normal fibres. It is due to a decrease of the stechiometric coefficient of Na+,K+-pump at denervation from 2.15 to 1.3 and to a change of the passive ionic permeability: at denervation permeability for Na+ increases from 0.52.10(-7) to 0.67.10(-7) cm.sec-1, and for K+ decreases from 0.75.10(-5) to 0.53.10(-5) cm.sec-1.     

Contributions to Physiology of the Antenna-Heart in Periplaneta americana (L.) (Blattodea: Blattidae)

An accessory pulsatile organ of an open circulatory system in insects supplying the antennae with haemolymph was investigated. The rhythm of this so-called antenna-heart is generated by a myogenic automatism and can be neuronally influenced via the nervus cardioantennalis.The action potentials of the muscle fibres show typical pre-depolarization and mostly no overshoot. A specific membrane resistance (R(m)) of about 660Omegacm(-2) was calculated for the fibres. Some electrical coupling between the muscle fibres is presumed for synchronization of any myogenically triggered heart beat which could actually be proved experimentally by current injection in the antenna-heart. However, intercalated disks or gap junctions could not be found. Nevertheless, a good coupling factor (U(2)/U(1)) between all fibres was demonstrated by parallel recordings and can be well described by a conductance model according to fibre topology.     

Recovery of isolated 'Na-rich' rat muscles in the presence of serum

The presence of serum in the recovery medium facilitates the restoration of intracellular Na and K concentrations and of the electrical properties in isolated ‘Na-Rich’ skeletal muscles (extensor digitorum longus) of K-deficient rats. The resting membrane potential to muscle fibres after recovery in the presence of serum closely agrees with that expected from the ionic theory based on steady state conditions.     

Effects of internal Na and external K concentrations on Na/K coupling of Na,K-pump in frog skeletal muscle

Summary To clarify the dependency of the Na/K coupling of the Na,K-pump on internal Na and external K concentrations in skeletal muscle, the ouabain-induced change in membrane potential, the ouabain-induced change in Na efflux and the membrane resistance were measured at various internal Na and external K concentrations in bullfrog sartorius muscle.Upon raising the internal Na concentration from 6 mmol/kg muscle water to 20 mmol/kg muscle water, the magnitude of the ouabain-induced change in membrane potential increased about eightfold and the magnitude of the ouabain-induced change in Na efflux increased about fivefold while the membrane resistance was not significantly changed. As the external K concentration increased from 1 to 10mm, the magnitude of the ouabain-induced change in membrane potential decreased (1/5.5 fold), while the magnitude of the ouabain-induced change in Na efflux increased (about 1.5-fold). The membrane resistance decreased upon raising the external K concentration from 1 to 10mm (1/2-fold). These observations imply that the values of the Na/K coupling of the Na,K-pump increases upon raising the internal Na concentration and decreases upon raising the external K concentration.     

Changes in electrical properties of muscle membrane systems during decoupling and recoupling induced by glycerol.

In isolated muscle fibres of the frog and of the crayfish the following electrical parameters were determined during the glycerol procedure from the voltage transients at 20 degrees C: the sarcoplasmic resistivity, Ri; the membrane resistance, Rm; the series tubular resistance, Rs; the surface membrane capacity, Cm; the tubular membrane capacity CT. No significant changes were found in fibres equilibrated with glycerol (G) saline. During the washout of glycerol only Ri and Cm remained unchanged. In reversibly decoupled crayfish fibres (300 mM-G) CT decreased to 70%, Rs increased to 175% and Rm increased to 200% of the control values. The changed parameters returned to control values upon reapplication of glycerol. In irreversibly decoupled fibres (500 and 600 mM-G) the changes in CT and Rs were more pronounced; and Rm was decreased. The resting potential remained constant with few mV. In frog fibres the changes in electrical parameters were in the same direction except the decrease of Rm during reversible decoupling (150 mM-G). The corresponding changes in reversible and irreversibly (300 mM-G) detubulated fibres were as follows: CT--60 (80) %; Rs--10 (14) times; Rm--50 (35) %.     

Electrical properties of skeletal muscle fibres of the flour moth larva Ephestia kuehniella

The electrical properties of the ventral longitudinal muscle fibres in the flour moth larva Ephestia kuehniella were investigated at rest and during electrical activity. The membrane resting potential was only partially dependent on the K-concentration gradient across the muscle membrane. The electrical constants λ, τ, R_m, R_i, and C_m were determined according to the equations for ‘short cables’ (Table 1). Current-voltage relationships of the muscle membrane were measured: they revealed anomalous as well as delayed rectification of the membrane. Stimulation of the muscle fibres with intracellular current pulses elicited graded action potentials in most fibres; in some fibres ‘all-or-none’ action potentials were generated. In contrast to graded action potentials these ‘all-or-none’ action potentials were propagated without decrement along the muscle fibre. Indirect stimulation of the muscle fibres resulted in large excitatory junction potentials which generally gave rise to action potentials.     

Inward rectifier potassium current in the frog embryonic skeletal myocytes

Pharmacological and kinetic properties of the inward rectifier potassium current Iir the frog embryonic skeletal myocytes were found to be identical to those of adult frog skeletal muscle fibres. The data obtained suggest that the Iir plays the main role in maintaining the myocytes resting membrane potential (RMP) when chloride conductance is insignificant. Changes of the integral conductance Gir and the RMP values correlated with the T-system development. The inward rectifier K+ channels, from the early stages of the muscle seem to be located in the T-tubule membranes.     

The effect of vanadate on the electrogenic Na+/K+ pump, intracellular Na+ concentration and electrophysiological characteristics of mouse skeletal muscle fibre

The present study reports a discrepancy between the effects of vanadate on the membrane Na+-K+-ATPase and the Na+/K+ pump of the skeletal muscle. Vanadate in concentration 4 X 10(-6) mol/l which is necessary to block the enzyme Na+-K+-ATPase activity of membrane fractions failed to inhibit the electrogenic Na+/K+ pump of intact muscle cells. The effect of vanadate on the electrophysiological parameters of the muscle fibre membrane required much higher vanadate levels, but again, Na+/K+ pump was still active. Vanadate in concentrations 4 X 10(-4) and 4 X 10(-5) mol/l depolarized the membrane potential and decreased the membrane resistance [apparently in consequence of enhanced passive membrane permeability for Na+ ions]. Action potentials and the electrical excitability of the muscle fibre membrane were reduced by these vanadate concentrations.