Single Frog Muscle Fibres Research Articles

Force-velocity relation in deuterium oxide-treated frog single muscle fibres during the rise of tension in an isometric tetanus.

1. The force-velocity (P-V) relation from a single fibres isolated from the semitendinosus muscle of the frog was determined at pre-set times during the rise of tension and the plateau of isometric tetani. The controlled-velocity release method was used. Experiments were performed at about 2.25 micrometers sarcomere length and at 3-4 degrees C or at 19-21 degrees C. 2. Replacing H2O with D2O resulted in a rapid large reduction of the peak twitch tension and of the speed of development of twitch and tetanic tensions. The tetanic tension (P0) was usually reduced, in certain fibres to as low as 5% of the value in H2O-Ringer solution. 3. The depression of twitch and tetanus characteristics was followed by a recovery, the duration of which varied greatly in different fibres. During the recovery period previous conditioning activity potentiated the tetanus characteristics. 4. After the end of the recovery period in D2O-Ringer solution both the peak twitch tension and the speed of development of tetanic tension was still greatly depressed, whereas the value of P0 was slightly greater than in H2O-Ringer. The speed of rise of isometric tension after a quick release imposed at the tetanus plateau was reduced in D2O-Ringer, usually to about 50% of the value in H2O-Ringer. 5. D2O increased the development time of the P-V relation and produced a conspicuous increase in the degree of its curvature. The value of V0 (the velocity of shortening at zero load) was not significantly depressed by D2O and it was the same independent both of the time after the beginning of stimulation and of the isometric tension at which the measurement was made. The P-V relation attained its final characteristics before the isometric tension reached the plateau. During the recovery period in D2O-Ringer, at the plateau of isometric tetani of different size, the relative force exerted at a given velocity of shortening was constant. 6. In D2O-treated fibres, NO3- and caffeine (i) potentiated the peak twitch tension and the speed of development of tetanic tension without affecting significantly the speed of the redevelopment of tension after a quick release imposed at the tetanus plateau and (ii) reduced the development time of the P-V relation, but did not affect either the degree of its curvature or the value of V0 and P0. 7. The results are discussed by assuming that the release of Ca2+ from the sarcoplasmic reticulum is a rate-limiting process for the development of activation and in turn for the development of isometric tension. In terms of the cross-bridge model of Huxley (1957), the time or Ca2+-dependent factor of activation appears to be the recruitment of actin sites for cross-bridge formation, whereas the value of the rate constants regulating the cross-bridge kinetics appears to be time and Ca2+-independent.

Light diffraction studies of active muscles fibres as a function of sarcomere length.

This investigation has established the following points. (1) Activation of single intact frog muscle fibres or mechanically skinned fibres results in a decrease in the intensity of the first-order line at all sarcomere lengths at which filament overlap is present. (2) At long sarcomere lengths (greater than 3.6 micrometers) the intensity decrease upon stimulation of intact fibres diminishes until above 3.9 micrometers no decrease is seen. In the skinned preparation, no intensity change is seen at sacromere lengths above 3.6 micrometers. (3) The intensity decrease seen in the intact fibres in the 3.6-3.9 micrometers sarcomere length region may be due to contraction of sarcomeres near the tendons, which have a shorter sarcomere length than those illuminated by the laser beam, and thus may not be stretched beyond filament overlap. (4) No intensity decrease is observed upon activation at very long (greater than 4.5 micrometers) sarcomere lengths. (5) Mechanically skinned fibres show a graded intensity response to free calcium. (6) Scans of first-order line width show no broadening upon activation, indicating that sarcomere length dispersion in the illuminated region of the fibre does not increase.

Calcium-dependent electrical activity and contraction of voltage-clamped frog single muscle fibres.

1. The electrical and mechanical activities of isolated frog muscle fibres have been simultaneously recorded under conditions which allow the development of a calcium permeability (chloride-free solution containing 72 mM-calcium levulinate instead of 115 mM-NaCl). 2. Long-lasting calcium action potentials and large and sustained contraction occur without inhibition of the potassium permeability. 3. The relationship observed between the slow inward calcium current and the amplitude of the contraction, under voltage-clamp conditions, resembles that found between IBa and tension in frog skeletal muscle and between ICa and the contraction in frog heart. A part of the mechanical response which is not abolished by manganese seems to be potential-dependent. 4. Integration of the current traces shows that the threshold of the contraction occurs at [Ca]1 near to 2.10(-6) M and that the contractile proteins are fully activated at [Ca]1 near to 10(-4) M, values which are in good agreement with those proposed by other authors. 5. The minimum calcium influx necessary to induce a detectable contraction is close to 4.8 p-moles/cm2. This value is 10-20 times larger than the calcium influx measured during a normal action potential. 6. On detubulated fibres the calcium permeability fails to develop: this indicates that this current originates from the T-system. The close correspondence between ICa and the contraction, and between the time course of the contraction at the end of the depolarizing steps suggests that the potential of the tubular membrane is better clamped than in normal physiological conditions.

Slow inward barium current and contraction on frog single muscle fibres.

1. Excitation-contraction coupling process in isolated frog muscle fibres, under conditions which allow the development of a Ba permeability, has been investigated by the simultaneous recording of electrical and mechanical activity. 2. The sustained contraction elicited by a long lasting Ba action potential depends on two mechanisms. The first is potential dependent, the second which is inhibited by MnCl2 (10 mM), depends on the inward flux of Ba ions. 3. The relationship observed between the inward IBa and the peak tension resembles that which has been observed between ICa and the contraction on other muscular structures. 4. The relative tension progressively declines as the intracellular Ba concentration increases and the contractility ends after a series of depolarizing pulses (or Ba action potentials). This indicates that the Ba ions which enter the cell release Ca ions and replace them in the intracellular storage sites. 5. Following a pretreatment with caffeine, the inward IBa fails to induce a contraction. Moreover a muscle which has been loaded with barium until the contraction ceases, does not develop a contracture in presence of caffeine. These results show that the Ba induced Ca release is located at the level of the sarcoplasmic reticulum. 6. Calculations show that the amount of Ba ions necessary to abolish the contractility corresponds to the maximum ability of the sarcoplasmic reticulum for Ca binding. 7. Almost all the inward flux of Ba ions and the contraction are abolished by glycerol-treatment which suggests that the coupling occurs at the T-system level. The results are discussed in regard to the technical limitations of the voltage-clamp method.

Depression of mechanical performance by active shortening during twitch and tetanus of vertebrate muscle fibres

Shortening during activity of frog single muscle fibres caused a graded depression of the contractile force that persisted for 800-900 ms during a partially fused or completely fused tetanus. The depression of force was not associated with a change of the shortening velocity at zero load. Passive shortening performed just before stimulation had no effect on the subsequent course of contraction. The decrease in isometric force produced by shortening was not significantly affected by a stretch applied immediately before or after the shortening was not significantly affected by a stretch applied immediately before or after the shortening phase. For a given amount of shortening the depressant effect during a fused tetanus was 8-28% of that produced during a twitch. The effect was substantially reduced, both during twitch and tetanus, in the presence of 0.5 mM caffeine. The length dependence of the movement effect was studied between 1.7 and 2.9 micrometer sarcomere spacing. Maximum depression of force (in per cent of the control at each length) was obtained at 2.1-2.2 micrometer sarcomere length, the effect being steadily reduced at shorter and more extended lengths. The Q10 of the depressant effect was 0.95 +/- 0.16 (S.D.). The features of the movement effect are consistent with a true deactivation of the contractile system as would occur if shortening reduced the binding of activator calcium to the regulatory proteins of the myofilaments.

Effect of glycerol treatment on sodium and potassium in isolated muscle fibres of the frog.

The sodium concentration in single frog muscle fibres vacuolated by glycerol treatment was significantly higher than in devacuolated fibres. Intracellular potassium concentration did not show any significant change. It is concluded that the transverse tubular system forms vacuoles with a high NaCl concentration upon glycerol removal.

The role of non-uniform sarcomere behaviour during relaxation of striated muscle.

It has previously been shown that there are marked non-uniformities in the pattern of sarcomere length changes during relaxation of a muscle fibre. This problem has now been studied in more detail during both twitch and tetanus in frog single muscle fibres and some of these results are presented to elucidate the mechanical consequences of the non-uniform sarcomere movements. The sarcomere behaviour during an isometric twitch is essentially the same as that observed during a tetanus in a given fibre: the sarcomere pattern is stable during the rising phase, the peak and the initial portion of the relaxation period. The pattern becomes non-uniform at a point (approx. 73% of peak force) which coincides with the beginning of a rapid fall in tension. At this point some segments (altogether c. 70% of the fibre length) start to shorten at the expense of others which are overextended. Maximum shortening, and elongation, is attained as the isometric tension approaches zero (2–5% of peak force). The majority of the sarcomeres in the fibre thus produce mechanical work (against a decaying load) during the isometric relaxation phase. The onset on non-uniformity varies with the time at which peak twitch force is attained. Thus, if a fibre is released to redevelop tension at different times during a twitch, the onset of non-uniformity is found to shift correspondingly to coincide with the beginning of the rapid tension fall in each case. The results suggest that the mechanical activity decays at different rates along the length of the fibre. The cross-bridges (reduced in number) in the weaker segment(s) will be stretched and when the stretch exceeds approximately 18 nm/half sarcomere the bridges start to slide. It appears that the yielding somehow further reduces the activity in this segment leading to a steeper decline in tension. It is possible to induce the non-uniform behaviour (and the rapid relaxation phase) at an earlier time by applying a stretch to the fibre so that the weak segments start to yield. In this way the rapid relaxation phase may be initiated already at the peak of the isometric twitch. The results suggest that the non-uniform behaviour provides a mechanism by which the isometric relaxation is speeded up. Evidence is presented to show that a similar mechanism may be effective also during relaxation of cardiac muscle.

Biphasic potassium contractures in frog single twitch muscle fibers and effects of various agents on the contractures.

The characteristics and the mechanism of the biphasic potassium contractures, i.e., an initial rapid contraction (initial component) and a slow secondary contraction (secondary component) were investigated in detail using single twitch fibers from the semitendinosus muscles of the frog, Rana japonica. The present experiments confirmed the results of CONSTANTIN (1971) and STEFANI and CHIARANDINI (1973) and further demonstrated that larger diameters (75--100 micron) of single fibers are required as one of the conditions for obtaining a biphasic potassium contracture. In addition, the effects of calcium removal from the external medium and the various agents, i.e., 2.5 microM dantrolene, 5 mM manganese ions, 7.5 mM procaine, 0.5 mM verapamil and 6.5 mM lincomycin on the initial and secondary components were examined. The pretreatment of a single fiber with calcium-free Ringer solution or with dantrolene or manganese ions mainly inhibited the secondary component, while it slightly or moderately inhibited the initial component in keeping with its steepness. In contrast, procaine, verapamil and lincomycin mainly inhibited the initial component, changing slightly or moderately the secondary component. It could be concluded, therefore, that the excitation-contraction coupling, i.e., the activation process of the initial component differs from that of the secondary component.

Birefringence signals and tension development in single frog muscle fibres at short stimulus intervals

The early large birefringence signal and mechanical activity were studied together in isolated single fibres of frog skeletal muscle with double stimulation at short stimulus intervals (2-60 msec) at room temperature and at 4--6 degrees C. In all fibres tested, extra tension and additional birefringence signal in response to the second stimulus appeared simultaneously and suddenly upon increasing the stimulus interval. The shape of the stimulus-interval versus tension-development curve makes it highly improbable that subthreshold calcium release occurs at shorter stimulus intervals; therefore, tension development reliably reflects Ca-release in these experiments. In contrast to the report by Suarez-Kurtz and Partker, birefringence signal and calcium release are shown not to be dissociated by double stimulation. This result supports the hypothesis that the early large birefringence signal is an intrinsic indicator of calcium release from the sr during EC-coupling in skeletal muscle.

Changes in diffraction patterns with length in single muscle fibres at rest.

The addition of a simple X--Y sampling circuit to a closed circuit television system (CCTV) permits measurements of a narrow profile of the laser diffraction patterns from single frog muscle fibres at rest. Results confirm that maximum intensity occurs at 2.95-3.00 micron, but a positive linear relationship between the dispersions of sarcomeres and sarcomere lengths from 2.7--3.8 micron is obtained in four isolated single fibres.

The origin of the instantaneous elasticity in single frog muscle fibres

High-speed cinematographic recordings of single tetanized muscle fibres during a quick decrease in length revealed that the shortening was mostly localized at the fibre segment nearest the released end of the fibre, indicating that the instantaneous elasticity may not originate from the elasticity of the cross-bridges.

Force‐velocity relation in normal and nitrate‐treated frog single muscle fibres during rise of tension in an isometric tetanus.

1. The force-velocity (P-V) relation for normal or NO-3 treated single fibres isolated from the semitendinosus muscle of the frog was determined at given times during the rise of tension and the plateau of isometric tetani. Experiments were made at about 2.25 micron sarcomere length and at constant temperatures, from 3 to 4.5 degrees C and from 19 to 21 degrees C. The controlled-velocity release method was used. 2. During the rise of tension, at any initial tension higher than about 0.2 P0, the lowest release velocity required to drop the tension to zero was the same as at the tetanic plateau, independent of the temperature and the presence of NO-3 ions in the bathing solution. 3. The degree of activation (measured by the steady force exerted at a given velocity of shortening lower than V0) increased with time, but attained its steady-state level before isometric tension. 4. At about 20 degrees C, frog muscle fibres at about 2.2 micron sarcomere length were only partially activated after a single stimulus. 5. NO-3 ions did not affect the steady-state P-V relation. At about 20 degrees C, NO-3 ions increased the rate of development of activation. Potentiation of the twitch contraction was due at least in part to this mechanism. 6. The 'relative' P-V relation appears to be independent of both the time after start of stimulation and the presence of NO-3 ions in the bathing solution. 7. The results are discussed in terms of the sliding filament model of Huxley (1957), assuming that either the number of actin sites available for cross-bridge formation, or the value of the rate constant for making of cross-bridges, is time dependent.

Metabolic correlates of fatigue and of recovery from fatigue in single frog muscle fibers.

Fatigue and recovery from fatigue were related to metabolism in single fibers of the frog semitendinosus muscle. The fibers were held at a sarcomere length of 2.3 microm in oxygenated Ringer solution at 15 degrees C and were stimulated for up to 150 s by a schedule of 10-s, 20-Hz tetanic trains that were interrupted by 1-s rest periods, after which they were rapidly frozen for biochemical analysis. Two kinds of fatigue were produced in relation to stimulus duration. A rapidly reversed fatigue occurred with stimulation for under 40 s and was evidenced by a decline in tetanic tension that could be overcome by 1 s of rest. A prolonged fatigue was caused by stimulation for 100-150 s. It was evidenced during stimulation by a fall in tetanic tension that could not be overcome by 1 s of rest, and after stimulation by a reduction, lasting for up to 82 min, in the peak tension of a 200-ms test tetanus. Fiber phosphocreatine (PCr) fell logarithmically in relation to stimulus duration, from a mean of 121 +/- 8 nmol/mg protein (SEM, n = 12) to 10% of this value after 150 s of stimulation. PCr returned to normal levels after 90-120 min of rest. Stimulation for 150 s did not significantly affect fiber glycogen and reduced fiber ATP by at most 15%. It is suggested that the prolonged fatigue caused by 100-150 s of tetanic stimulation was caused by long-lasting failure of excitation-contraction coupling, as it was not accompanied by depletion of energy stores in the form of ATP. One possibility is that H+ accumulated in fatigued fibers so as to interfere with the action of Ca2+ in the coupling process.

Voltage‐clamp experiments on frog single skeletal muscle fibres: evidence for a tubular sodium current.

1. A late inward current following the initial sodium current is sometimes observed on frog single skeletal muscle fibres studied in voltage-clamp conditions by the double sucrose-gap method. 2. The late inward current is time and voltage dependent; it is sensitive to extracellular sodium concentration and inhibited by tetrodotoxin. Therefore, it is very probably carried by sodium ions. 3. It is assumed that the late current reflects a regenerative increase of sodium conductance in the tubular membrane. The main argument favouring this assumption is that the late current is never observed on detubulated fibres. 4. Inactivation studies show a marked difference between the evolution of the two inward currents when the membrane resting potential is increased by previous hyperpolarizations in a high range of values: the late current is decreased while the initial current remains constant. The decrease of the late current is suppressed by tetraethylammonium ions or in a potassium-free medium. On detubulated fibres, the sodium current is never decreased by conditioning hyperpolarizations. 5. It is postulated that a fast transient potassium current, located in the bubular membrane, should develop contemporaneously with the sodium current. 6. The decay of the sodium inactivation is anaylsed on semilogarithmic plot: on normal fibres, it runs in two phases while on detubulated fibres, one phase only is found.

Fatigue and posttetanic potentiation in single muscle fibers of the frog

At 15 degrees C, direct stimulation of frog single muscle fibers at a frequency of 20 Hz produced a tetanic tension that remained constant for 20 s and then declined. The decline was reversed during 1-s interruptions of the stimulus train in the first 50 s of stimulation, but not with longer stimulation. Posttetanic potentiation (PTP), characterized by prolonged twitch relaxation and contraction times and elevation of twitch height, remained for 10-40 min after a 10-s tetanus and for at least 90 min after a 50- to 150-s tetanus. Posttetanic fatigue appeared only after at least 50s of tetanic stimulation. Fatigue was manifested invariably by a reduction in the height of a 200-ms tetanic contraction and usually by a reduction in twitch height after PTP. Fatigued fibers recovered normal contractile responses in 40-160 min. Hypertonic solutions, which blocked contraction in response to tetanic stimulation, prevented posttetanic fatigue but not PTP. The observations suggest that fatigue is caused by a failure in excitation-contraction coupling, probably in relation to consumption of metabolic substrates. Even 10-s tetani which do not produce fatigue can affect muscle contractile function for up to 40 min.

Birefringence signals from surface and t‐system membranes of frog single muscle fibres.

1. When the tonicity of Ringer is increased above 2-5 times normal and a single fibre stimulated externally, the large, early birefringence signal preceding twitch tension (Baylor & Oetliker, 1975, 1977 a,b) is sufficiently reduced and delayed so as to reveal a small but distinct signal ('1st component") preceding it. For an average-sized fibre, the deltaI/I of the 1st component was (minus) 1 to 2 x 10-5. 2. The time course of the 1st component superimposed with the surface action potential simultaneously recorded by an internal micro-electrode. The polarity of the 1st component reversed with compensation. 3. From these characteristics, the 1st component is thought to arise from a small change in optical retardation of the surface membrane due to the action potential. 4. When a fibre was impaled with two micro-electrodes, retardation changes accompanying small hyperpolarizing and depolarizing current steps were detected. In some cases the polarity of the observed signal was opposite in sign to that expected for a retardation change only from the surface membrane. 5. Because the anatomical orientation of the T-system appears to be primarily transverse rather than longitudinal, these signals of opposite polarity are probably, on balance, due to retardation changes from the membranes of the T-system. 6. The possible origin of the large birefringence signal preceding contraction is discussed.

Contractile inactivation in frog single denervated muscle fibers

After preliminary conditioning depolarizations, single muscle fibers of the frog were tested for ability to contract in response to depolarization by 100 mM K+ Ringer solution. Denervated fibers (6-42 days) lose their ability to produce a 100 mM K+ contracture more rapidly than do control fibers. This decrease in 100 mM K+ contracture size (inactivation) is dependent on length of exposure to and magnitude of the conditioning depolarization and on the calcium concentration in the external medium. At 0.4 mM Ca++, the inactivation is 3 times faster than at 1.5 mM Ca++. The rate of contracture loss is not correlated with fiber diameter or the number of days after failure of neuromuscular transmission, and the preliminary conditioning depolarizations do not affect the rate of terminal relaxation from the 100 mM K+ contractures.

Longitudinal impedance of single frog muscle fibers.

The longitudinal impedance of single skeletal muscle fibers has been measured from1 to 10,000 Hz in an oil gap apparatus which forces current to flow longitudinally down the fiber. The impedance observed is purely resistive in some fibers from the semitendinosus muscle and in two fibers from the sartorius muscle. In other fibers from the semitendinosus muscle a small phase shift is observed. The mean value of the maximum phase shift observed from all fibers is 1.07 degrees. The artifacts associated with the apparatus and method are examined theoretically and it is shown that one of the likely artifacts could account for the small phase observed. It is concluded that the longitudinal impedance of skeletal muscle fibers is essentially resistive and that little, if any, longitudinal current crosses the membranes of the sarcoplasmic reticulum.

Fatigue in frog single muscle fibres.

WHEN a whole striated muscle is stimulated repetitively, its contractile force decreases and it is said to be fatigued1–3. Fatigue does not occur in dystrophic muscles, and is characteristic of fast but not slow mammalian and amphibian muscle fibres4,5. The complexity of the excitation–contraction process in fast muscle fibres has made it difficult to determine when fatigue sets in. Restoration of twitch tension of fatigued fibres by caffeine6,7 suggests that fatigue is a failure of calcium release, although the depletion, during prolonged contraction, of necessary energy-rich metabolites8,9 could be responsible for fatigue. Contraction can be uncoupled from calcium release and excitation by hypertonic solutions (refs 10–12 and unpublished results of S. R. Taylor, R. Rudel and J. R. Blinks). We have now found that hypertonic uncoupling prevents fatigue, so that fatigue must be linked to contraction. We have also found that single muscle fibres can recover after being fatigued by prolonged tetanisation.

Potassium contractures of frog single denervated muscle fibers: time course and central spread.

ARTICLESPotassium contractures of frog single denervated muscle fibers: time course and central spreadSC Stuesse, BD Lindley, and AC KirbySC Stuesse, BD Lindley, and AC KirbyPublished Online:01 Jul 1974https://doi.org/10.1152/ajplegacy.1974.227.1.200MoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByDenervated frog skeletal musclePfl�gers Archiv European Journal of Physiology, Vol. 400, No. 3Caffeine contractures in denervated frog musclePfl�gers Archiv European Journal of Physiology, Vol. 400, No. 3Changes in sensitivity of muscle spindles of fast and slowly contracting muscles after chronic de-efferentationNeurophysiology, Vol. 13, No. 2THE ROLE OF EXTRACELLULAR CALCIUM IN THE CONTRACTIONS PRODUCED BY ACETYLCHOLINE IN CHRONICALLY DENERVATED MUSCLE19 July 2012 | British Journal of Pharmacology, Vol. 61, No. 4Denervated frog skeletal muscle: Calcium content and kinetics of exchangeExperimental Neurology, Vol. 53, No. 1 More from this issue > Volume 227Issue 1July 1974Pages 200-208 Copyright & PermissionsCopyright © 1974 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1974.227.1.200PubMed4546363History Published online 1 July 1974 Published in print 1 July 1974 Metrics