Action Potential in the Transverse Tubules and Its Role in the Activation of Skeletal Muscle

The double sucrose-gap method was applied to single muscle fibers of Xenopus. From the "artificial node" of the fiber, action potentials were recorded under current-clamping condition together with twitches of the node. The action potentials were stored on magnetic tape. The node was then made inexcitable by tetrodotoxin or by a sodium-free solution, and the wave form of the action potential stored on magnetic tape was imposed on the node under voltage-clamp condition (simulated AP). The twitch height caused by the simulated AP's was always smaller than the twitch height produced by the real action potentials, the ratio being about 0.3 at room temperature. The results strongly suggest that the transverse tubular system is excitable and is necessary for the full activation of twitch, and that the action potential of the tubules contributes to about 70 % of the total mechanical output of the normal isotonic twitch at 20°C. Similar results were obtained in the case of tetanic contraction. At a temperature near 10°C, twitches produced by the simulated AP were not very different (85 % of control amplitude) from the twitches caused by real action potentials. This indicates that the excitability of the tubules becomes less necessary for the full activation of twitch as the temperature becomes lower.     

An Assessment of the Double Sucrose-Gap Voltage Clamp Technique as Applied to Frog Atrial Muscle

The homogeneity of voltage clamp control in small bundles of frog atrial tissue under double sucrose-gap voltage clamp conditions was assessed by intracellular microelectrode potential measurements from cells in the test node region. The microelectrode potential measurements demonstrated that (1) good voltage control of the impaled cell existed in the absence of the excitatory inward currents (e.g., during small depolarizing clamp pulses of 10-15 mV), (2) voltage control of the impaled cell was lost during either the fast or slow excitatory inward currents, and (3) voltage control of the impaled cell was regained following the inward excitatory currents. Under nonvoltage clamp conditions the transgap recorded action potential had a magnitude and waveform similar to the intracellular microelectrode recorded action potentials from cells in the test node. Transgap impedance measured with a sine-wave voltage of 1,000 Hz was about 63% of that measured either by a sine-wave voltage of 10 Hz or by an action potential method used to determine the longitudinal resistance through the sucrose-gap region. The action potential data in conjunction with the impedance data indicate that the extracellular resistance (R_s) through the sucrose gap is very large with respect to the longitudinal intracellular resistance (R_i); the frequency dependence of the transgap impedance suggests that at least part of the intracellular resistance is paralleled by a capacitance. The severe loss of spatial voltage control during the excitatory inward current raises serious doubts concerning the use of the double sucrose-gap technique to voltage clamp frog atrial muscle.     

The Effect of Shortening on the Time-Course of Active State Decay

The active state describes the force developed in a muscle when the contractile elements are neither lengthening nor shortening. Recently it was suggested that perturbations used to measure the active state also alter the time-course of the active state. The present research was undertaken to assess quantitatively the effect of two such perturbations, isotonic shortening and quick release, on the active state in frog sartorius muscle. Methods were developed which allowed the determination of active state points following periods of controlled isotonic shortening or quick release early in the contraction cycle. All experiments were carried out within the plateau region of the length-tension curve. Both isotonic shortening and quick release altered the active state decay. The active state force decreased as the extent of shortening or release was increased. For each 0.1 mm of isotonic shortening there was a 2% decrease in active state force. Quick release produced a larger decrement. From this data we conclude that the time-course of active state can be measured only in relative terms because it is altered by the motion which takes place in the contractile machine while the active state is being measured. This finding helps to resolve paradoxes in the literature relating to the time-course of the active state, calculated and experimentally determined isometric tetanic myograms, and the heat of shortening.     

Muscle Volume Changes

Measurements have been made of the volume changes accompanying single isometric and isotonic twitches of frog sartorius muscle. The volume change consists of a rapid increase, a subsequent decrease, and a return to the initial volume; the order of magnitude of increase and decrease is 10^-5 cc/g of muscle. This volume change is length-dependent: the initial increase becomes more pronounced as the initial length of the muscle is decreased, while the volume decrease is greatest at reference length and is diminished for longer and shorter initial lengths. Muscle volume changes are also dependent upon temperature and amount of shortening: the return phase is prolonged as the temperature is lowered; and, in an isotonic twitch, a volume increase accompanying muscle shortening is superimposed upon the volume change described for an isometric twitch. These "shortening volume changes" may correspond to the volume decrease observed in frog muscle under a passive stretch. If the active state is prolonged by the use of a frog Ringer solution in which iodide ions have been substituted for chloride ions, the time course of the volume decrease is likewise prolonged; this suggests a relationship between the volume decrease and the active state of the muscle.     

Membrane Potentials of the Lobster Giant Axon Obtained by Use of the Sucrose-Gap Technique

A method similar to the sucrose-gap technique introduced be Stäpfli is described for measuring membrane potential and current in singly lobster giant axons (diameter about 100 micra). The isotonic sucrose solution used to perfuse the gaps raises the external leakage resistance so that the recorded potential is only about 5 per cent less than the actual membrane potential. However, the resting potential of an axon in the sucrose-gap arrangement is increased 20 to 60 mv over that recorded by a conventional micropipette electrode when the entire axon is bathed in sea water. A complete explanation for this effect has not been discovered. The relation between resting potential and external potassium and sodium ion concentrations shows that potassium carries most of the current in a depolarized axon in the sucrose-gap arrangement, but that near the resting potential other ions make significant contributions. Lowering the external chloride concentration decreases the resting potential. Varying the concentration of the sucrose solution has little effect. A study of the impedance changes associated with the action potential shows that the membrane resistance decreases to a minimum at the peak of the spike and returns to near its initial value before repolarization is complete (a normal lobster giant axon action potential does not have an undershoot). Action potentials recorded simultaneously by the sucrose-gap technique and by micropipette electrodes are practically superposable.     

Feedback in the Contractile Mechanism of the Frog Heart

Shortening causes a transient decrease, extension an increase, in activity during contractures of the frog ventricle induced by high Ca or by isosmotic K solution. This is shown by the fact that, after the immediate passive shortening, the muscle is extended under isotonic conditions when the load is diminished, and that under isometric conditions quick release causes first a rapid drop, then a further, much slower, fall of tension. Increasing the load or stretching induce the opposite effects. At low temperatures all rapid changes in length produce oscillations of low frequency. These responses are due to a sensitive feedback mechanism similar to that previously demonstrated for insect fibrillar muscle. That this mechanism comes into play in the heart under normal conditions and controls the time-course of the twitch is demonstrated by the observation that relaxation begins earlier the greater the shortening. Thus, during afterloaded isotonic twitches the onset of relaxation is advanced as the load is diminished.     

Mechanical properties of denervated amphibian muscle

Denervated amphibian muscle does not show the prolongation of action potential found in mammalian denervated muscle. It was, therefore, predicted that denervated amphibian muscle would not show prolongation of the mechanical twitch. The sartorius muscles in one leg of toads--Xenopus borealis--were denervated for 140-268 days. Isometric twitch time to peak, time to half relaxation and twitch/tetanus ratio were not changed following denervation, confirming our prediction. Twitch tension decreased to 68% and tetanic tension decreased to 75% of control values. The maximum velocity of unloaded shortening (muscle length/s) was also unchanged.     

Effects of K+ on the twitch and tetanic contraction in the sartorius muscle of the frog, Rana pipiens. Implication for fatigue in vivo.

The effects of increasing the extracellular K+ concentration on the capacity to generate action potentials and to contract were tested on unfatigued muscle fibers isolated from frog sartorius muscle. The goal of this study was to investigate further the role of K+ in muscle fatigue by testing whether an increased extracellular K+ concentration in unfatigued muscle fibers causes a decrease in force similar to the decrease observed during fatigue. Resting and action potentials were measured with conventional microelectrodes. Twitch and tetanic force was elicited by field stimulation. At pHo (extracellular pH) 7.8 and 3 mmol K+.L-1 (control), the mean resting potential was -86.6 +/- 1.7 mV (mean +/- SEM) and the mean overshoot of the action potential was 5.6 +/- 2.5 mV. An increased K+ concentration from 3 to 8.0 mmol.L-1 depolarized the sarcolemma to -72.2 +/- 1.4 mV, abolished the overshoot as the peak potential during an action potential was -12.0 +/- 3.9 mV, potentiated the twitch force by 48.0 +/- 5.7%, but did not affect the tetanic force (maximum force) and the ability to maintain a constant force during the plateau phase of a tetanus. An increase to 10 mmol K+.L-1 depolarized the sarcolemma to -70.1 +/- 1.7 mV and caused large decreases in twitch (31.6 +/- 26.1%) and tetanic (74.6 +/- 12.1%) force. Between 3 and 9 mmol K+.L-1, the effects of K+ at pHo 7.2 (a pHo mimicking the change in interstitial pH during fatigue) and 6.4 (a pHo known to inhibit force recovery following fatigue) on resting and action potentials as well as on the twitch and tetanic force were similar to those at pHo 7.8. Above 9 mmol K+.L-1 significant differences were found in the effect of K+ between pHo 7.8 and 7.2 or 6.4. In general, the decrease in peak action potential and twitch and tetanic force occurred at higher K+ concentrations as the pHo was more acidic. The results obtained in this study do not support the hypothesis that an accumulation of K+ at the surface of the sarcolemma is sufficiently large to suppress force development during fatigue. The possibility that the K+ concentration in the T tubules reaches the critical K+ concentration necessary to cause a failure of the excitation-contraction coupling mechanism is discussed.     

Effect of temperature on the inward rectifier and gramicidin A-induced channels in the membrane of frog skeletal muscle fibres

The conductance of frog skeletal muscle fibres in isotonic K2SO4 solution has been measured. Experiments were carried out under current-clamp conditions using a double sucrose-gap technique. The potassium conductances of the inward rectifier and the gramicidin channel in the same muscle fibre were compared. Potassium conductance of the inward rectifier increased with the temperature, with a value of Q10 1.55 +/- 0.09 (n = 8) under hyperpolarization, and Q10 2.38 +/- 0.23 (n = 6) for the depolarizing stimulus, the difference between Q10 of potassium and gramicidin channels being statistically insignificant.     

Loss of the plateau of the cardiac action potential in hypertonic solutions

The effect of hypertonicity on the electrical properties of vertebrate myocardial cells was studied in ventricular muscle fibers of guinea pig, cat, frog, and chicken. The latter two species do not have a T-tubule system, whereas the former two do. In hypertonic solutions (2 x isotonic) produced by addition of sucrose or excess of NaCl, cell diameter decreased and there was a slight hyperpolarization and decrease in action potential overshoot. In guinea pig and cat, the hypertonic solution caused a decrease in input resistance and the plateau of the action potential to disappear in some of the cells; contractions of the entire ventricle also became depressed. These effects were reversed by returning the muscle fibers to isotonic solution. Addition of 5 mM SrCl₂ to the hypertonic solution also caused the plateau component and contraction to reappear. In frog and chick cells, loss of the plateau component and contraction never occurred in hypertonic solution, and input resistance increased. Urea and glycerol hyperosmolarity (2 x) caused no loss of the plateau component or contraction. If the frog and chicken ventricular, and guinea pig atrial myocardial cells (all of which lack T tubules) were to serve as an adequate control for possible effects of hypertonicity on the surface membrane and on contractile proteins, then the results suggest that swelling of the T tubules of mammalian myocardial cells leads to loss of the plateau component.     

Opposite effects of cooling on twitch contractions of skeletal muscle isolated from tropical toads (Leptodactylidae) and northern frogs (Ranidae)

Cooling increases the twitch force of frog skeletal muscle (Rana temporaria; Rana pipiens), but decreases the twitch force of tropical toad muscle (Leptodactylus insularis). Action potentials and intramembranous charge movement in frog and toad fibers were slowed identically by cooling. Cooling increased the integral of twitch Ca²⁺ detected by aequorin in frog fibers (1.4-fold), while also decreasing the peak and slowing the rate of decay. Conversely, cooling decreased the integral (0.6-fold) and the peak of twitch Ca²⁺ in toad fibers, without affecting the rate of decay. The difference in entire Ca²⁺ transients may account for cold-induced twitch potentiation in frogs and twitch paralysis in toads. In sustained contractions of toad fibers, cooling markedly decreased maximum force caused by: (i) tetanic stimulation, (ii) two-microelectrode voltage clamp steps, (iii) high [K⁺], or (iv) caffeine. Maximum force in sustained contractions was decreased moderately by cooling frog fibers. Rapid rewarming and simultaneous removal of high [K⁺] or caffeine during a sustained contraction, caused toad muscle force to rise towards the value corresponding to the warm temperature. This did not occur after removing high [K⁺] or caffeine from toad fibers kept in the cold. Transmission electron micrographs showed no relevant structural differences. Parvalbumins are thought to promote relaxation of frog muscle in the cold. The unique parvalbumin isoforms in toad muscle apparently lack this property. Accepted: 27 August 1998     

K+-induced twitch potentiation is not due to longer action potential

The objective of this study was to determine whether an increased duration of the action potential contributes to the K+-induced twitch potentiation at 37 degrees C. Twitch contractions were elicited by field stimulation, and action potentials were measured with conventional microelectrodes. For mouse extensor digitorum longus (EDL) muscle, twitch force was greater at 7-13 mM K+ than at 4.7 mM (control). For soleus muscle, twitch force potentiation was observed between 7 and 11 mM K+. Time to peak and half-relaxation time were not affected by the increase in extracellular K+ concentration in EDL muscle, whereas both parameters became significantly longer in soleus muscle. Decrease in overshoot and prolongation of the action potential duration observed at 9 and 11 mM K+ were mimicked when muscles were respectively exposed to 25 and 50 nM tetrodotoxin (TTX; used to partially block Na+ channels). Despite similar action potentials, twitch force was not potentiated by TTX. It is therefore suggested that the K+-induced potentiation of the twitch in EDL muscle is not due to a prolongation of the action potential and contraction time, whereas a longer contraction, especially the relaxation phase, may contribute to the potentiation in soleus muscle.     

Energetics of Shortening Muscles in Twitches and Tetanic Contractions : II. Force-Determined Shortening Heat

The extra heat liberation accompanying muscular shortening, the force-determined shortening heat, is defined as the difference between the heat produced when shortening occurs and that produced in an isometric contraction developing the same amount of force and performing the same amount of internal work. Based on this definition, the initial energy production in twitches and tetanic contractions (E) is given by E = A + f (P, t) + α_Fx + W, where A is the activation heat, f(P, t), the tension-related heat (a heat production associated with the development and maintenance of tension), α_Fx, the force-determined shortening heat, and W, the external work. It is demonstrated that this equation accurately accounts for the time-course of heat evolution and the total initial energy production in both twitches and tetani at 0°C. The force-determined shortening heat is liberated, during shortening, in direct proportion to (a) the distance shortened, and (b) the force against which shortening occurs. The normalized value of the force-determined shortening heat coefficient, α_F/P_o, is the same in both the twitch and the tetanus. Finally, this formulation of the muscle's energy production also accounts for the total energy production in afterload isotonic twitches at 20°C, where a Fenn effect is not demonstrable.     

Velocity transients and viscoelastic resistance to active shortening in cat papillary muscle.

When isotonic force steps were applied to activated papillary muscles, the velocity was almost never constant. Early rapid shortening associated with the step persisted for 2-7 ms after the step ends. The early rapid shortening is attributed to lightly damped series elastic recoil and velocity transients of the contractile elements. In most steps, the subsequent velocity declines progressively with shortening, and most of the decline in velocity can be accounted for by compression of a viscoelastic element in parallel with the contractile elements. To demonstrate this, the time course of isotonic velocity was compared with a model in which the force-velocity characteristics of the contractile element were assumed to be constant, and the decline in velocity was due to increasing compression of the viscoelastic element. This model predicted the observed results except that the predicted velocities rose progressively above the measured values for steps to light loads applied late in the twitch, and fell below the velocity trace for heavy loads applied early in the twitch. These deviations would occur if rapid shortening caused deactivation late in the twitch, and if activation were rising early in the twitch. A conditioning step applied to the muscle during the rise of force depressed both isometric force and maximum velocity measured at the peak of force; isometric force was more depressed with later conditioning steps than with earlier steps, while maximum velocity was depressed by about the same extent with both early and late steps. This difference between the effects on isometric force and maximum velocity are explained by a combination of deactivation and viscoelastic load.     

Effects of (+) and (-) enantiomers of calcium channel agonist, Bay K 8644, on mechanical and electrical responses of frog skeletal muscle

The effects of (+) and (-) enantiomers of Bay K 8644, a Ca2+ channel agonist, on the mechanical and electrical properties of frog skeletal muscle fibers were investigated. In the concentration range of 10(-6) to 10(-5) M, both (+) and (-) enantiomers of Bay K 8644 significantly increased the maximum amplitudes of twitch responses. Both (+) and (-) enantiomers of Bay K 8644, at higher concentrations such as 10(-4) M, greatly depressed the amplitudes of twitches. Potentiating and depressing effects of (-) enantiomer of Bay K 8644 on twitch responses were significantly greater than those of the (+) enantiomer. At all concentrations used, both (+) and (-) enantiomers of Bay K 8644 significantly decreased the area under the tetanic force x time curve. In intracellular recordings, it was found that the depressing effects of both (+) and (-)-Bay K 8644 on tetanic contractions and twitch responses were due to the inhibition of action potentials. The inhibitory effect of (-) enantiomer of Bay K 8644 on action potentials also was significantly greater than that of the (+) enantiomer. In conclusion, present results suggest that, in contrast with cardiac muscle fibers, (+) and (-) enantiomers of Bay K 8644 have similar inhibitory effects on the electrical and mechanical properties of frog skeletal muscle fibers.     

The effect of pressure on the lateral swimming muscle of the European eel Anguilla anguilla and the deep sea eel Histiobranchus bathybius; results of Challenger cruise 6B/85

1. The viability of Histiobranchus lateral muscle was prolonged up to 7 times by recompression of the tissue. 2. The maximum twitch contraction force of both Anguilla and Histiobranchus was recorded at a pressure between 150 and 350 atm. At 1 atm Anguilla developed 60% maximum force and Histiobranchus 10-20% maximum force. 3. Twitch contraction time doubled for a pressure increase of 400 atm. This effect is predicted to halve the maximum swimming speed at 4000 m and is discussed in relation to muscle force and anaerobic support.     

A phenomenological model for estimating metabolic energy consumption in muscle contraction

A phenomenological model for muscle energy consumption was developed and used in conjunction with a simple Hill-type model for muscle contraction. The model was used to address two questions. First, can an empirical model of muscle energetics accurately represent the total energetic behavior of frog muscle in isometric, isotonic, and isokinetic contractions? And second, how does such a model perform in a large-scale, multiple-muscle model of human walking? Four simulations were conducted with frog sartorius muscle under full excitation: an isometric contraction, a set of isotonic contractions with the muscle shortening a constant distance under various applied loads, a set of isotonic contractions with the muscle shortening over various distances under a constant load, and an isokinetic contraction in lengthening. The model calculations were evaluated against results of similar thermal in vitro experiments performed on frog sartorius muscle. The energetics model was then incorporated into a large-scale, multiple-muscle model of the human body for the purpose of predicting energy consumption during normal walking. The total energy estimated by the model accurately reflected the observed experimental behavior of frog muscle for an isometric contraction. The model also accurately reproduced the experimental behavior of frog muscle heat production under isotonic shortening and isokinetic lengthening conditions. The estimated rate of metabolic energy consumption for walking was 29% higher than the value typically obtained from gait measurements.     

Slowing of velocity during isotonic shortening in single isolated smooth muscle cells. Evidence for an internal load

In single smooth muscle cells, shortening velocity slows continuously during the course of an isotonic (fixed force) contraction (Warshaw, D.M. 1987. J. Gen. Physiol. 89:771-789). To distinguish among several possible explanations for this slowing, single smooth muscle cells were isolated from the gastric muscularis of the toad (Bufo marinus) and attached to an ultrasensitive force transducer and a length displacement device. Cells were stimulated electrically and produced maximum stress of 144 mN/mm2. Cell force was then reduced to and maintained at preset fractions of maximum, and cell shortening was allowed to occur. Cell stiffness, a measure of relative numbers of attached crossbridges, was measured during isotonic shortening by imposing 50-Hz sinusoidal force oscillations. Continuous slowing of shortening velocity was observed during isotonic shortening at all force levels. This slowing was not related to the time after the onset of stimulation or due to reduced isometric force generating capacity. Stiffness did not change significantly over the course of an isotonic shortening response, suggesting that the observed slowing was not the result of reduced numbers of cycling crossbridges. Furthermore, isotonic shortening velocity was better described as a function of the extent of shortening than as a function of the time after the onset of the release. Therefore, we propose that slowing during isotonic shortening in single isolated smooth muscle cells is the result of an internal load that opposes shortening and increases as cell length decreases.     

Caffeine- and Potassium-Induced Contractures of Frog Striated Muscle Fibers in Hypertonic Solutions

The effect of hypertonic solutions on the caffeine- and KCl-induced contractures of isolated fibers of frog skeletal muscle was tested. Hypertonic solutions, twice the normal osmotic strength, prepared by adding NaCl or sucrose, potentiate the caffeine-induced contractures. The fibers may develop tensions of 3.6 kg/cm² of fiber transverse section. The same hypertonic medium reduced the peak tension of KCl-induced contractures. Thus the hypertonic condition does not affect the contractile mechanism itself. These findings give further support to the view that the differential effect of hypertonic solution is on the excitation-contraction coupling mechanism. Extracellular calcium is not essentially required for the first few of a series of caffeine-induced contractures either in hypertonic or in isotonic solutions.     

Fatigue and recovery of dynamic and steady-state performance in frog skeletal muscle

Muscle fatigue reflects alterations of both activation and cross-bridge function, which will have markedly different affects on steady-state vs. dynamic performance. Such differences offer insight into the specific origins of fatigue, its mechanical manifestation, and its consequences for animal movement. These were inferred using dynamic contractions (twitches and cyclic work as might occur during locomotion) and steady-state performance with maximal, sustained activation (tetani, stiffness, and isokinetic force) during fatigue and then recovery of frog (Rana pipiens) anterior tibialis muscle. Stiffness remained unaltered during early fatigue of force and then declined only 25% as force dropped 50%, suggesting a decline with fatigue in first the force-generating ability and then the number of cross bridges. The relationship between stiffness and force was different during fatigue and recovery; thus the number of cross bridges and force per cross bridge are not intimately linked. Twitch duration increased with fatigue and then recovered, with trajectories that were remarkably similar to and linear with changes in tetanic force, perhaps belying a common mechanism. Twitch force increased and then returned to resting levels during fatigue, reflecting a slowing of activation kinetics and a decline in cross-bridge number and force. Net cyclic work fatigued to the degree of becoming negative when tetanic force had declined only 15%. Steady-state isokinetic force (i.e., shortening work) declined by 75%, while cyclic shortening work declined only 30%. Slowed activation kinetics were again responsible, augmenting cyclic shortening work but greatly augmenting lengthening work (reducing net work). Steady-state measures can thus seriously mislead regarding muscle performance in an animal during fatigue.