Structural changes in single muscle fibers after stimulation at a low frequency (original) (raw)
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Ultrastructural changes accompanying development of fatigue in frog twitch skeletal muscle fibres
Endocrine regulations, 2005
The aim of the present study was to characterise and compare alterations in the ultrastructure of the functionally identified isolated twitch skeletal muscle fibres of the frog after repeated tetanic stimulation and under experimental conditions which modified their fatigability. Single isolated twitch muscle fibres of m. iliofibularis of adult frogs Rana temporaria were subjected to intermittent tetanic stimulation. Fibres at specified degree of fatigue were processed for electron microscopic observation and ultrastructural examination. The fatigue-resistant (FR) fibres that developed 90% of the control tetanic tension after 10 min stimulation in ordinary Ringer's solution showed regions with dilated intermyofibrillar spaces containing small vesicles and swollen mitochondria. In addition to the changes observed in FR fibres, the easily fatigued (EF) fibres that produced 50% of the original tension after 3 min stimulation showed small vacuoles in the sarcoplasm. In EF fibres tha...
Correlated Morphological and Physiological Studies on Isolated Single Muscle Fibers
The Journal of Cell Biology, 1968
Living muscle fibers of crayfish become dark during efflux of Cl-. This change in appearance is correlated with occurrence of vacuolation in the fixed fibers. The vacuoles begin at and are mainly confined to the terminals of the transverse tubular system (TTS) which are in diadic contact with the sarcoplasmic reticulum (SR). In electron micrographs swellings more than 1 µ in diameter may be seen connected to the sarcolemma or sarcolemmal invaginations by relatively unswollen tubules about 300–500 A wide. Darkening of the living fibers can be reversed by causing an influx of Cl-. Vacuoles are then absent in the fixed preparations. These findings accord with the conclusion that the membrane of the TTS is anion permselective. Localization of the selectivity to the membrane of the terminals of the TTS strengthens the hypothesis that a channeling of current flow is responsible for initiation of excitation-contraction coupling. During the swelling, and upon its reversal, the area of the m...
Pfl�gers Archiv European Journal of Physiology, 1997
The mechanical and energy characteristics of isolated fast-twitch muscle fibres (type 1) of Xenopus laevis in isometric-and isovelocity contractions were measured at 20°C. The fibres were stimulated at either 60 Hz or 20 Hz to produce contractions at different levels of activation. The high stimulation frequency gave fused contractions, while at the low stimulation frequency tension fluctuated. When maximum isometric force had been reached, the fibres were shortened by 10% of the fibre length at different velocities. At 60 Hz stimulation during shortening the rate of heat production increased above the isometric rate of heat production. At 20 Hz stimulation during shortening, however, the rate of heat production was not different from the isometric rate of heat production. Mechanical efficiency was the same at the high and low level of activation. The actomyosin efficiency (i.e. the mechanical efficiency corrected for "activation heat") was highest at the low level of activation. We conclude that in fast-twitch muscle fibres from X. laevis, actomyosin efficiency is highest for partially activated muscle. From a comparison of the present results with those obtained from a study of slow-twitch muscle fibres presented earlier, it is concluded that fasttwitch muscle fibres are less efficient than slow-twitch muscle fibres.
Twitch and Tetanic Tension during Culture of Mature Xenopus laevis Single Muscle Fibres
Archives Of Physiology And Biochemistry, 2001
Investigation of the mechanisms of muscle adaptation requires independent control of the regulating factors. The aim of the present study was to develop a serum-free medium to culture mature single muscle fibres of Xenopus laevis. As an example, we used the culture system to study adaptation of twitch and tetanic force characteristics, number of sarcomeres in series and fibre cross-section. Fibres dissected from m. iliofibularis (n = 10) were kept in culture at a fibre mean sarcomere length of 2.3 mm in a culture medium without serum. Twitch and tetanic tension were determined daily. Before and after culture the number of sarcomeres was determined by laser diffraction and fibre cross-sectional area (CSA) was determined by microscopy. For five fibres twitch tension increased during culture and tetanic tension was stable for periods varying from 8 to 14 days ('stable fibres'), after which fibres were removed from culture for analysis. Fibre CSA and the number of sarcomeres in series remained constant during culture. Five other fibres showed a substantial reduction in twitch and tetanic tension within the first five days of culture ('unstable fibres'). After 7-9 days of culture, three of these fibres died. For two of the unstable fibres, after the substantial force reduction, twitch and tetanic tension increased again. Finally at day 14 and 18 of culture, respectively, the tensions attained values higher than their original values. For stable fibres, twitch contraction time, twitch half-relaxation time and tetanus 10%-relaxation time increased during culture. For unstable fibres these parameters fluctuated. For all fibres the stimulus threshold fluctuated during the first two days, and then remained constant, even for the fibres that were cultured for at least two weeks. It is concluded that the present culture system for mature muscle fibres allows long-term studies within a well-defined medium. Unfortunately, initial tetanic and twitch force are poor predictors of the long-term behaviour of the fibres.
The Histochemical Journal, 1989
Intensities of histochemical and immunohistochemical reactions in muscle fibres of Rana and Xenopus have been estimated microphotometrically, and the data from serial sections statistically analysed. Quantitative validities of reactions and measurements have also been assessed against independent published evidence. It is concluded that NADH-tetrazolium reductase overestimates tonic-fibre aerobic capacities and the actomyosin ATPase reaction overestimates their contraction speeds. However, it appears that succinate dehydrogenase, despite being a near-equilibrium enzyme of particulate distribution, indicates the relative aerobic capacities of fibres with acceptable accuracy when lightly reacted. Capacities for aerobic and anaerobic metabolism are positively correlated over all types of fibre (r typically-0.6 for 200 fibres), perhaps as an adaptation to environmental hypoxia. Multivariate clusters (indicating fibre types) have been sought, using Ward's method with optimizing procedures (iterative relocation and multivariate-normal modelling). Cluster analysis confirms the subjective identifications of two 'slow/tonic' types in Xenopus (labelled T5 and $4) but of only one (T5) in Rana. Division of the 'fast family' twitch fibres into three types (F1 F3) in both genera, with metabolic capacity related inversely to apparent shortening velocity, is highly supportable by objective criteria. However, statistically significant subdivisions also present themselves. Rana F2 and Xenopus F1 clusters can be bisected according to metabolic capacity; and Xenopus F2 fibres fall into three subtypes reflecting different isomyosin contents. In the different types of twitch fibre, ratios of myofibrillar ATP consumption rate to aerobic capacity increase up to 30-fold with contraction speed, but anaerobic/aerobic ratios do so only 5-fold.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2005
Chronic low-frequency stimulation of rabbit tibialis anterior muscle over a 24-h period induces a conspicuous loss of isometric tension that is unrelated to muscle energy metabolism (J.A. Cadefau, J. Parra, R. Cusso, G. Heine, D. Pette, Responses of fatigable and fatigueresistant fibres of rabbit muscle to low-frequency stimulation, Pflugers Arch. 424 (1993) 529-537). To assess the involvement of sarcoplasmic reticulum and transverse tubular system in this force impairment, we isolated microsomal fractions from stimulated and control (contralateral, unstimulated) muscles on discontinuous sucrose gradients (27-32-34-38-45%, wt/wt). All the fractions were characterized in terms of calcium content, Ca 2+ /Mg 2+ -ATPase activity, and radioligand binding of [ 3 H]-PN 200-110 and [ 3 H]ryanodine, specific to dihydropyridine-sensitive calcium channels and ryanodine receptors, respectively. Gradient fractions of muscles stimulated for 24 h underwent acute changes in the pattern of protein bands. First, light fractions from longitudinal sarcoplasmic reticulum, enriched in Ca 2+ -ATPase activity, R 1 and R 2 , were greatly reduced (67% and 51%, respectively); this reduction was reflected in protein yield of crude microsomal fractions prior to gradient loading (25%). Second, heavy fractions from the sarcoplasmic reticulum were modified, and part (52%) of the R 3 fraction was shifted to the R 4 fraction, which appeared as a thick, clotted band. Quantification of [ 3 H]-PN 200-110 and [ 3 H]ryanodine binding revealed co-migration of terminal cisternae and t-tubules from R 3 to R 4 , indicating the presence of triads. This density change may be associated with calcium overload of the sarcoplasmic reticulum, since total calcium rose three-to fourfold in stimulated muscle homogenates. These changes correlate well with ultrastructural damage to longitudinal sarcoplasmic reticulum and swelling of ttubules revealed by electron microscopy. The ultrastructural changes observed here reflect exercise-induced damage of membrane systems that might severely compromise muscle function. Since this process is reversible, we suggest that it may be part of a physiological response to fatigue. D
Slow and fast fatigable frog muscle fibres: electrophysiological and histochemical characteristics
General physiology and biophysics, 2005
Continuous activity of isolated frog gastrocnemius muscle fibres provoked by repetitive stimulation of 5 Hz was used as an experimental model for fatigue development in different fibre types. Parameter changes of the elicited intracellular action potentials and mechanical twitches during the period of uninterrupted activity were used as criteria for fatigue evaluation. Slow fatigable muscle fibre (SMF) and fast fatigable muscle fibre (FMF) types were distinguished depending on the duration of their uninterrupted activity, which was significantly longer in SMFs than in FMFs. The normalized changes of action potential amplitude and duration were significantly smaller in FMFs than in SMFs. The average twitch force and velocity of contraction and relaxation were significantly higher in FMFs than in SMFs. Myosin ATPase (mATPase) and succinate dehydrogenase activity were studied by histochemical assessment in order to validate the fibre type classification based on their electrophysiologi...
Action Potential in the Transverse Tubules and Its Role in the Activation of Skeletal Muscle
The Journal of General Physiology, 1974
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 tet...
Frog skeletal muscle fibers recovering from fatigue have reduced charge movement
2000
Following prolonged exercise, muscle force production is often impaired. One possible cause of this force de®cit is impaired intracellular activation. We have used single skeletal muscle ®bers from the lumbrical muscle of Xenopus laevis to study the eects of fatigue on excitation±contraction coupling. Fatigue was induced in 13 intact ®bers. Five ®bers recovered in normal Ringer only (fatigued-only ®bers). The remaining eight ®bers were subjected to a brief hypotonic treatment (F-H ®bers) that is known to prolong the eects of fatigue. Intramembrane charge movement, changes in intracellular calcium concentration ([Ca 2+ ] i ) and force transients were measured in a single Vaseline gap chamber under voltage clamp. In F-H ®bers, membrane capacitance was reduced. Confocal microscopy showed that this was not due to closure of the transverse tubules. The amount of normalized intramembrane charge was reduced from 21.0 2.8 nC/lF (n = 10) in rested ®bers to 12.2 1.1 nC/lF in F-H ®bers. However, the voltage dependence of intramembrane charge movement was unchanged. In F-H ®bers, force production was virtually abolished. This was the consequence of the greatly reduced [Ca 2+ ] i accompanying a depolarizing pulse. In recovering fatigued-only ®bers, while the maximal available charge was not signi®cantly smaller (18.3 1.1 nC/ lF), both calcium and force were reduced, albeit to a lesser extent than in F-H ®bers. The data are consistent with a model where fatigue reduces the number of voltage sensors in the t-tubules and, in addition, alters the coupling between the remaining functional voltage sensors and the calcium channels of the sarcoplasmic reticulum.
The Journal of Physiology, 2003
Mammalian skeletal muscle fibres are generally rich in mitochondria, which make up about 10-15 % of a fibre's volume (Eisenberg, 1983; Chen et al. 2001). Mitochondria are distributed differentially in skeletal muscle, with a higher density being found close to the sarcolemma than deeper in the fibre (Eisenberg, 1983; Nakae et al. 1999). The key function of mitochondria has always been thought to be that of an energy supplier that adapts readily to the changing demands of the working muscle. Energy production by the mitochondria increases during exercise. It is generally accepted that increases in metabolites such as ADP and inorganic phosphate act as potent stimuli to increase ATP generation by the mitochondria (e.g. Vendelin et al. 2000). The nature of Ca 2+ involvement in the increased metabolic activity of the mitochondria is not entirely understood. Several mitochondrial dehydrogenases involved in the tricarboxylic acid cycle are known to be sensitive to Ca 2+ (Denton & McCormack, 1990; McCormack et al. 1990) and recent experiments suggest that Ca 2+ directly stimulates mitochondrial respiration (Kavanagh et al. 2000). Aside from their important role as energy suppliers, mitochondria may play a further role, which is to modulate the cytosolic free [Ca 2+ ] ([Ca 2+ ] i), as has been demonstrated to occur in neurones (David et al. 1998), cardiac myocytes (Duchen et al. 1998) and in frog muscle fibres (Lännergren et al. 2001). Sembrowich et al. (1985) demonstrated that mitochondria isolated from fast-and slow-twitch mammalian skeletal muscle have the capacity to take up Ca 2+ , and the kinetic characteristics of that Ca 2+ uptake suggested that, in the intact fibre, mitochondria could play a significant role in lowering [Ca 2+ ] i in slowtwitch muscle. Gillis (1997) demonstrated that pharmacological inhibition of mitochondrial Ca 2+ uptake with ruthenium red results in slowed force relaxation in skinned fibres. In addition, several groups have reported that mitochondria isolated from skeletal muscle after exhaustive exercise contain more Ca 2+ than those isolated from non-exercised muscle (Duan et al. 1990; Madsen et al. 1996), although such accumulation appeared to depend on the mode of exhaustive exercise (Tate et al. 1980). One factor that may contribute to the decline in force during a