Transmission phenomena and early tension recovery in skinned muscle fibres of the frog

Tension responses due to small rapid length changes completed in 50 s were obtained from segments with different length of single fibres of the ileofibularis muscle of the frog. The very early parts of the responses varied with segment length. A simulation of the early parts of the response was carried out by means of a linear model in which the fibre is regarded as a rod of infinitesimally small segments containing undamped elasticity, damped elasticity and mass in series. In the simulation corrections were included for the effects caused by the viscosity and density of the surrounding fluid and for the force transducer characteristics. The results indicate the presence of a very rapid component in the fast recovery with a time constant of 5–15 s. The undamped elasticity of the activated fibres corrected for their passive properties was such that a sudden shortening corresponding to 2.6 nm/half sarcomere would reduce active tension to zero.     

Tension responses to rapid length changes in skinned muscle fibres of the frog

Tension responses due to rapid length changes completed in 50 and 150 s were obtained from activated skinned single fibres of the ileofibularis muscle of the frog. The natural frequency of the force transducer was about 50 kHz. The length changes ranged between –1% and +0.5% of the fibre segment length. The sarcomere length was adjusted to 2.15 m. The temperature was maintained at 2.5° C. The transmission velocity estimated from these recordings obtained on fibre segments with different length was 230 m/s in fully activated segments and 112 m/s in relaxed segments. The initial part of the responses during the length changes consisted of an abrupt change in tension reaching an extreme value T₁, which depended on the amplitude as well as the duration of the length change. A partial rapid recovery towards a plateau occurred after the length change. The reciprocal half-time of this recovery increased with the amplitude of the displacement both for stretches as well as releases up to about 5 nm/half sarcomere.     

Calcium sensitivity and myofibrillar protein isoforms of rat skinned skeletal muscle fibres

We investigated the calcium sensitivity for tension generation of different fibre types and the possible correlation between calcium sensitivity and the presence of distinct regulatory protein and myosin light chain (MLC) isoforms in rat skinned skeletal muscle fibres. Fibre types 1, 2A and 2B were identified by electrophoretic analysis of myosin heavy chain (MHC) isoforms. Fibres showing more than one MHC isoform were discarded. Type 1 fibres from the soleus showed a higher pCa (–log₁₀ [Ca], where [ ] denotes concentration) threshold and a lower slope of pCa/tension curve than type 2 extensor digitorum longus (EDL) fibres; between type 2 fibres, type 2B showed the higher slope of pCa/tension curve. Type 1 fibres from different muscles showed similar calcium sensitivities when containing only the slow set of regulatory proteins and MLC; when both slow and fast isoforms were present, calcium sensitivity shifted toward fast type fibre values. Type 2A fibres from different muscles showed a similar calcium sensitivity, independently of the set (purely fast or mixed) of regulatory proteins and MLC. It is suggested that when both fast and slow isoforms of regulatory proteins and of MLC are present in a muscle fibre, calcium sensitivity is dictated mainly by the fast isoforms.     

Endurance exercise effects on the contractile properties of single,skinned skeletal muscle fibres of young rats

Single fibres were isolated from the extensor digitorum longus (EDL) and the soleus (SOL) muscles of the hindlimb from young male Wistar rats which had undergone a 10-week programme of endurance swimming from the age of 2 weeks. Fibres were mechanically skinned and activated with Ca²⁺- and Sr²⁺ -buffered solutions. Muscle fibres were classified by means of well-defined criteria concerning various aspects of their contractile behaviour. Most fibres could be allocated into specific groups; however, a significant proportion (13% of the sampled population) did not fit these rigid classifications but displayed contractile activation characteristics common to more than one fibre type. In these cases models which used a combination of both fast- and slow-twitch contractile and regulatory properties were used to characterise the activation behaviour of fibres. It is proposed that the exercise, initiated at a young age, induced changes in the contractile characteristics of the single fibres by modifying protein isoforms of the contractile apparatus.     

Alteration of calcium sensitivity of skinned frog skeletal muscle fibres by inositol triphosphate and calmodulin antagonists

We investigated the influence of inositol triphosphate (IP₃), trifluoperazine (TFP), and perhexiline on the calcium sensitivity of freeze-dried frog semitendinosus muscle fibres. Further, the effect of IP₃ on calcium release from the sarcoplasmic reticulum (SR) of frog semitendinosus fibres skinned by saponin was studied. IP₃ decreased the calcium sensitivity of freeze-dried frog skeletal muscle fibres and failed to induce a calcium release from SR of saponin-skinned fibres. Freeze-dried frog skeletal muscle fibres were strongly sensitized for calcium by TFP and perhexiline.     

Tension development and calcium sensitivity in skinned muscle fibres of the frog

Calcium activated isometric tension development was measured in single skinned muscle fibres of the ileofibularis muscle of the frog. The experiments were carried out at 5°C, pH=6.9, 1 mM free Mg²⁺ and an ionic strength of 160 mM. A Hill curve was fitted to the isometrically developed tension at different Ca²⁺ concentrations by means of a non-linear least mean square approximation. At a sarcomere length of 2.15 m, the Ca²⁺ concentration for half maximum tension (K) was 1.6 M. This Ca²⁺ concentration decreased with increasing sarcomere length; at 2.7 m, K was 1.1 M and at 3.1 m, K was 0.9 M. Therefore, Ca sensitivity is increased at larger sarcomere lengths. Consequently, the optimal sarcomere length for tension development shifted to larger values when the Ca²⁺ concentration was lowered. Osmotic compression of the fibre at 2.15 m by means of 5% Dextran also caused an increase in Ca sensitivity (K was 1.0 M). At 2.7 m, addition of 5% Dextran hardly affected the Ca sensitivity. The possible role of the interfilament spacing in the explanation of these results discussed.     

The relationship between ATP hydrolysis and active force in compressed and swollen skinned muscle fibers of the rabbit

We investigated whether the inhibition of force generation observed in compressed muscle fibers is accompanied by a coupled reduction in hydrolytic activity. Isometric force and rates of ATP hydrolysis (ATPase) were measured as functions of the relative width of chemically skinned skeletal muscle fiber segments immersed in relaxing (pCa>8) and activating (pCa 4.9) salt solutions. Osmotic radial compression of the fiber segment was produced (with little or no affect on striation spacing) by adding Dextran T500 to the bathing media. ADP as a product of ATP hydrolysis in fibers undergoing 10–15 min contractions was measured using high pressure liquid chromatography. Compression of the (initially swollen) fiber segment with dextran produced a slight (4%) increase in average active force and then, with further compression, a sharp decrease (with maximum around in situ width). With compression, the average ATPase of the fiber decreased monotonically, and with extreme compression (with 0.22 g dextran per ml), ATPase fell to a fifth of its level determined in dextran-free solution while force was abolished. The time course of active force development was described by the sum of two exponential functions, the faster of which characterized the rate of rise. Fiber compression (0.14 g dextran per ml) reduced the rate of rise of force ten-fold compared to that in dextran-free solution. Hindrance of cross movement is proposed to account for the inhibition of active force generation and (coupled) ATPase in compressed fibers.     

Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle

Thapsigargin has been reported to inhibit ATP-dependent Ca²⁺ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca²⁺ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca²⁺ content of the SR. The SR was first depleted of Ca²⁺ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 M was required to inhibit Ca²⁺ loading by 50%. In contrast, another SR Ca²⁺ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 M and total inhibition at 50 M. When cyclopiazonic acid (100 M) was applied after, rather than during, Ca²⁺ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 M), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca²⁺ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.     

Rate of isometric tension development in relation to calcium binding of skinned muscle fibres

Isolated muscle fibres from the sartorius or semitendinosus muscles of frogs were mechanically skinned and kept in a relaxed state in a medium containing Mg-ATP and EGTA. When subjected to a rapid increase in internal calcium ion concentration tension rose relatively slowly in comparison to the time course of establishment of the new calcium concentration. Stiffness measurements made during the rise of tension yielded the same stiffness to tension ratio as that observed at steady state force. The linear force extension curve of the activated fibres (T₁-curves) measured at various moments during the rise of tension extrapolated to zero tension intersected the base line at the same length (–0.8% L_o). This suggests that the extent of myosin interaction increases with the same time course as tension.The rate of tension development accompanying a Ca-jump was strongly increased by an increase in calcium ion concentration and there was a linear relationship between the logarithm of the rate tension development and pCa. The rate of recovery of tension following a large quick release > 2% L_o was not calcium sensitive, and occurred at a rate more than an order of magnitude faster than the corresponding calcium activation in the range of pCa's studied. We suggest that the slowness of tension development accompanying a rapid calcium activation reflects slow reactions occurring after a single Ca-ion has bound to a myofilament binding site and does not reflect the slowness of actin and myosin interaction.This work was supported by a grant from the Deutsche Forschungs-gemeinschaft Gu 160/3. One of us (P.J.G.) was supported by the Royal Society European Science Exchange Programme     

Inhibition of force production in compressed skinned muscle fibers of the frog

Calcium-activated force development in skinned frog muscle fibers is inhibited by osmotically compressing the fiber, probably owing to a decrease in spacing between the myofilaments. This inhibition depends upon sarcomere length in that fibers at long lengths must be compressed further than those at short lengths to achieve the same degree of inhibition. As a result, this length dependency of inhibition tends to compensate for the reduction of force due solely to the decrease in interfilament spacing which occurs with stretch in intact fibers.     

The contribution of the sarcoplasmic reticulum Ca2+-transport ATPase to caffeine-induced Ca2+ transients of murine skinned skeletal muscle fibres

The present study was carried out to investigate the contribution of the Ca²⁺-transport ATPase of the sarcoplasmic reticulum (SR) to caffeine-induced Ca²⁺ release in skinned skeletal muscle fibres. Chemically skinned fibres of balb-C-mouse EDL (extensor digitorum longus) were exposed for 1 min to a free Ca²⁺ concentration of 0.36 μM to load the SR with Ca²⁺. Release of Ca²⁺ from the SR was induced by 30 mM caffeine and recorded as an isometric force transient. For every preparation a pCa/force relationship was constructed, where pCa = −log₁₀ [Ca²⁺]. In a new experimental approach, we used the pCa/force relationship to transform each force transient directly into a Ca²⁺ transient. The calculated Ca²⁺ transients were fitted by a double exponential function: Y ₀ + A ₁⋅exp (−t/t ₁) + A ₂⋅exp(t/t ₂), with A ₁ < 0 < A ₂, t ₁ < t ₂ and Y ₀, A ₁, A ₂ in micromolar. Ca²⁺ transients in the presence of the SR Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) were compared to those obtained in the absence of the drug. We found that inhibition of the SR Ca²⁺-ATPase during caffeine-induced Ca²⁺ release causes an increase in the peak Ca²⁺ concentration in comparison to the control transients. Increasing CPA concentrations prolonged the time-to-peak in a dose-dependent manner, following a Hill curve with a half-maximal value of 6.5 ± 3 μM CPA and a Hill slope of 1.1 ± 0.2, saturating at 100 μM. The effects of CPA could be simulated by an extended three-compartment model representing the SR, the myofilament space and the external bathing solution. In terms of this model, the SR Ca²⁺-ATPase influences the Ca²⁺ gradient across the SR membrane in particular during the early stages of the Ca²⁺ transient, whereas the subsequent relaxation is governed by diffusional loss of Ca²⁺ into the bathing solution. Received: 2 February 1996/Accepted: 1 April 1996     

Cross-bridge stiffness in Ca2+-activated skinned single muscle fibres

Tension transients, in response to small and rapid length changes (completed within 40 s), were obtained from skinned single frog muscle fibres incubated in activating solutions with varying concentrations of Ca²⁺. The first 2 ms of these transients were described by a linear model in which the fibre is regarded as a rod composed of infinitesimally small, identical segments containing a mass, one undamped elastic element and in the case of relaxed fibres two damped elastic elements in series, or in the case of activated fibres three such elastic elements in series. The stiffness of activated fibres, expressed in elastic constants or apparent elastic constants, increased with increasing concentrations of Ca²⁺. All the damped elastic constants that were necessary to describe the tension responses of activated fibres were proportional to isometric tension. However, the undamped elastic constant did not increase linearly with increasing isometric tension. Equatorial X-ray diffraction patterns were obtained from single frog muscle fibres under similar conditions as under which the tension transients were obtained. The filament spacing (d ₁₀)of Ca²⁺-activated single frog muscle fibres decreased with increasing isometric force, whereas the intensity ratio (I ₁₁/I₁₀)increased linearly with increasing isometric force. From experiments in which dextran (MW 200000 Da) was added, it followed that such a change in filament spacing would modify passive stiffness. The d ₁₀value of relaxed fibres decreased and stiffness increased with increasing concentrations of the polymer dextran, whereas I ₁₁/I₁₀remained constant. The relation of stiffness and filament spacing with concentration of dextran was used to eliminate the effect of decreased filament spacing on stiffness of activated fibres. After correction for changes in filament spacing the undamped complicance C ₁, normalized to tension, was not constant, but increased with increasing isometric tension. If we assume that isometric tension is proportional to the number of force generating cross-bridges, this means that only part of the undamped complicance of activated fibres is located in the crossbridges.     

Sprint-training effects on some contractile properties of single skinned human muscle fibres

The effects of sprint training on the contractile properties of human muscle fibres obtained by needle biopsy were investigated. Individual fibres were mechanically skinned and activated by Ca²⁺- and Sr²⁺-buffered solutions at pH 7.1, and allocated to distinct populations on the basis of their contractile characteristics. The majority of fibres sampled pre-training could be separated into the three major fibre groups: Populations I (24/70, 34%), II (25/70, 36%) and III (18/70, 26%), which exhibited characteristics similar to those of histochemically classified type I, IIA and IIB fibres, respectively. The remainder (3/70, 4%) represented another fibre group, with intermediate characteristics. The muscle fibres were also activated by Ca²⁺ at a reduced pH of 6.6, to mimic the intracellular acidification that occurs during intense exercise. Lowering pH increased the threshold for contraction by Ca²⁺, reduced Ca²⁺ sensitivity, and increased the steepness of the force-pCa relationship, in all fibres sampled from the three major fibre groups. Maximum force was not significantly reduced in any fibre population. In the post-training sample, the three major fibre types were present in different proportions: Populations I (10/52, 19%), II (20/52, 38.5%) and III (11/52, 21%). Three other fibre groups sampled in low numbers exhibited contractile characteristics intermediate between Population I and Population II. Following sprint training all of the three main fibre populations exhibited higher thresholds for contraction by, and lower sensitivities to, Sr²⁺ but not Ca²⁺, compared with the fibres sampled pre-training. Maximum force was significantly lower in Population II fibres after sprint training. At pH 6.6, post-trained Population III fibres exhibited even lower Ca²⁺ sensitivity, with concomitant increases in the threshold for contraction and force-pCa curve steepness.     

Shortening velocity and force/pCa relationship in skinned crab muscle fibres of different types

Single fibres of three different types, which had been characterized histochemically with regard to differences in myofibrillar adenosine triphosphatase (ATPase) activity and its pH stability, were microdissected from freeze dried preparations of the closer muscle in walking legs of the crab Eriphia spinifrons. Shortening velocities were determined in slack tests and under constant load conditions in maximally Ca²⁺-activated skinned muscle fibres. Force/pCa relationships were also measured for the different types of fibres. Compared with data on vertebrate muscles, all crab muscle fibres required large length changes to reach zero force and showed low Ca²⁺ sensitivity for isometric force generation. The length/time relationship obtained from slack tests had a biphasic course. Maximal velocity of filament sliding differed in the three types of fibres investigated. The filament sliding of type IV fibres was about 3 times faster than that of type I fibres. The values obtained for type II fibres ranged in between. These data are positively correlated with myofibrillar ATPase activity determined histochemically. Ca²⁺ sensitivity of force generation was lowest in the fast type IV fibres. It was high in the slow type I and the faster contracting type II fibres. Ca²⁺ sensitivity in crab muscle seems not to be correlated with speed of shortening.     

Compared properties of the contractile system of skinned slow and fast rat muscle fibres

Chemically skinned fibres from soleus and plantaris rat muscles were used to compare the contractile properties of slow and fast muscles. The maximal isometric tension appeared larger in plantaris than in soleus fibres. The apparent Ca²⁺ threshold for activation was lower in slow than in fast fibres while Ca²⁺ concentrations required to obtain either the maximal tension or half maximal tension (pCa₅₀) were lower in fast than in slow fibres. This apparent difference in Ca²⁺ sensitivity will be discussed. As could be expected from other studies, a faster force development in plantaris than in soleus fibres occurred. However, one interesting new result showed that in soleus, the kinetics of the tension development estimated by the t _max parameter were slightly dependent on the Ca²⁺ concentration whereas the t ₅₀ parameter changed significantly with the Ca²⁺ concentration. In plantaris, both t _max and t ₅₀ parameters were found to depend strongly on the Ca²⁺ concentration. Finally, the plantaris muscle showed a greater caffeine sensitivity than the soleus muscle. All the results suggested that the Ca-regulatory mechanism in the slow fibres was essentially different from that in the fast fibres.     

Inhibition of TnI-TnC interaction and contraction of skinned muscle fibres by the synthetic peptide TnI [104–115]

Circular dichroism was used to study the induction of helix in TnC or TnI-TnC by the TnI peptide [104–115] at various Ca²⁺ concentrations. The increase in negative ellipticity and pCa²⁺ values for the peptide-TnC complex, indicates that binding of the peptide to TnC, induces a small helical conformational change in TnC. This results in an increase in the Ca²⁺ binding constant and the pCa₅₀ value required to induce 50% of Ca²⁺-dependent helix in TnC. The introduction of the peptide to a preformed mixture of TnI-TnC resulted in an increase in negative ellipticity and a decrease in the pCa₅₀ and the apparent Ca²⁺ binding constant towards the values obtained for the TnI peptide-TnC complex and away from those of TnI-TnC. This demonstrates that the TnI peptide can successfully compete with TnI for TnC and thereby inhibit the TnI-TnC interaction. The addition of the TnI peptide to skinned rabbit psoas or porcine cardiac fibres resulted in the inhibition of the force development and a decrease in the pCa₅₀ values required for 50% Ca²⁺ activation. The magnitude of the inhibition of tension development and the shift in the Ca²⁺ sensitivity for skinned cardiac muscle fibres was approximately half that observed with skeletal muscle fibres. In view of the CD findings, these skinned fibre results can be accounted for by the peptide inhibiting the TnI interaction with TnC. However, it is possible that the TnI peptide also has a direct inhibitory effect on TM-actin. Mastoparan, another TnC binding peptide, also inhibited the tension development in skinned skeletal and cardiac muscle fibres, but was much less efficient than the TnI peptide.Abbreviations used Boc N-tert-butyloxycarbonyl - Tn troponin - TM tropomyosin - TnI troponin I - TnC troponin C - HPLC high performance liquid chromatography - DIEA diisopropylethylamine - TFA trifluoroacetic acid - DCC dicyclohexylcarbodiimide - 2-Cl-Z 2-chlorobenzyloxycarbonyl - Tos 4-toluenesulfonyl - EGTA ethylene glycol bis(-amino ethyl ether) N,N,N,N-tetraacctic acid - DTE 1,2-ethanedithiol - ME -mercaptoethanol - S1 myosin subfragment 1 - acto-S1 actin and myosin subfragment This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie, and by grants from the Medical Research Council of Canada, the Alberta Heart Foundation and a studentship (J. V. E.) from the Alberta Heritage Foundation for Medical Research     

Short-range elasticity after tetanic stimulation in single muscle fibres of the frog

The time course of the stiffness during the relaxation period following tetanic stimulation was studied in isolated single muscle fibres of the frog. The stiffness was measured by subjecting the fibres to a sudden stretch at constant velocity from an initial sarcomere length of 2.2 μm, and related to the tension carried by the muscle fibre immediately before the stretch. Both stiffness and tension fell close to the resting level during the first second (4°C) after the end of stimulation. While stiffness and tension fell proportionally during the phase of almost exponential decline in tension following the shoulder in the tension recording, the relative drop in stiffness was less than the relative drop in tension during the preceding phase of almost linear decline in tension corresponding to the first 300–400 ms after the end of stimulation.     

Series elastic properties of skinned muscle fibres in contraction and rigor

Summary Isometric tension of skinned fibres from the frog semitendinosus muscle is sigmoidally related to Ca²⁺ concentration betweenpCa 7 and 6. Stiffness measurements showed that the Ca²⁺-activated tension may be due to recruitment of attached cross-bridges. In the absence of ATP (rigor solution) the skinned fibre develops a rigor tension which reaches about 80–110% of the maximum Ca²⁺-activated tension.However, stiffness measurements showed that in rigor many more cross-bridges are attached to actin at any one moment than in contraction. It was concluded that the force per cross-bridge is 37% smaller in rigor than in contraction.     

Shortening induced deactivation of skinned fibres of frog and mouse striated muscle

The depressant effect of active shortening, previously established in intact muscle fibres, was studied during calcium induced contractures of chemically skinned fibres from the semitendinosus muscle of Rana temporaria and the psoas muscle of the mouse. The decrease in contractile activity was determined by comparing the rate of force redevelopment (at a given tension level) after a large (test) and a small (control) release step. Under standard experimental conditions (ionic strength: frog 135 mM, mouse 190 mM; Ca²⁺ 3.0 μM; Mg²⁺: frog 25 μM, mouse 100 μM; MgATP^2-: frog 1.0 mM, mouse 2.0 mM) active shortening of 0.15 μm per sarcomere (in excess of control release) reduced the contractile activity by approximately 50% of the control in both frog and mouse muscle fibres. Full contractile activity was regained within 4 s during isometric activity after the shortening phase. The depressant effect of shortening was steadily reduced, to almost complete disappearance of the effect, by increasing the free calcium concentration within the range 1.5–12.0 μM. Similarly, an increase in ionic strength from 105 to 235 mM reduced the depressant effect by approximately 40%. In contrast, there was a progressive enhancement of the shortening effect as the magnesium ion concentration was increased from 25 to 590 μM. It is proposed that interaction between the myosin cross-bridges and the thin filament during sarcomere shortening leads to a decrease in troponin-calcium binding resulting in a temporary deactivation of the contractile system.     

The effect of exercise on the contractile properties of single skinned fast- and slow-twitch skeletal muscle fibres from the adult rat

The effects of long-term endurance exercise on the contractile properties of single skinned muscle fibres from adult rats, were investigated. Adult (4-month-old) male rats were subjected to a 16-week, high-intensity endurance swimming programme, where animals carried a load (corresponding to 2% of body wt), during all 2-h training sessions. At the conclusion of the training period, muscle fibres isolated from the extensor digitorum longus (EDL), and soleus (SOL), could be classified into distinct classes or fibre types on the basis of their Ca²⁺- and Sr²⁺-activated contractile characteristics. The fast-twitch EDL comprised two fibre populations, while the slow-twitch SOL was found to be composed of three distinct fibre types. Endurance swimming modified the contractile characteristics of fibres from both the EDL and SOL, but exerted greater influence on those of the SOL. This was illustrated by significant increases in the sensitivity to Ca²⁺ and Sr²⁺, and a lower threshold for contraction by these activating ions, in the exercised group. Not one of the total of 272 fibres sampled, exhibited mixed fast- and slow-twitch contractile characteristics, often associated with exercise-induced fibre type transformations. Thus, high-intensity endurance swimming induced changes in some single muscle fibre contractile properties of adult rats, but did not cause major changes in fibre type distribution.