Influence of ionic strength on the time course of force development and phosphate release by dogfish muscle fibres

We measured the effects of ionic strength (IS), 200 (standard) and 400 mmol l⁻¹ (high), on force and ATP hydrolysis during isometric contractions of permeabilized white fibres from dogfish myotomal muscle at their physiological temperature, 12°C. One goal was to test the validity of our kinetic scheme that accounts for energy release, work production and ATP hydrolysis. Fibres were activated by flash photolysis of the P ³-1-(2 nitrophenyl) ethyl ester of ATP (NPE-caged ATP), and time-resolved phosphate (P_i) release was detected with the fluorescent protein MDCC-PBP, N -(2[1-maleimidyl]ethyl)-7-diethylamino-coumarin-3-carboxamide phosphate binding protein. High IS slowed the transition from rest to contraction, but as the fibres approached the isometric force plateau they showed little IS sensitivity. By 0.5 s of contraction, the force and the rate of P_i release at standard and high IS values were not significantly different. A five-step reaction mechanism was used to account for the observed time courses of force and P_i release in all conditions explored here. Only the rate constants for reactions of ATP, ADP and P_i with the contractile proteins varied with IS, thus suggesting that the actin–myosin interactions are largely non-ionic. Our reaction scheme also fits previous results for intact fibres.     

Actomyosin energy turnover declines while force remains constant during isometric muscle contraction

Energy turnover was measured during isometric contractions of intact and Triton-permeabilized white fibres from dogfish ( Scyliorhinus canicula ) at 12°C. Heat + work from actomyosin in intact fibres was determined from the dependence of heat + work output on filament overlap. Inorganic phosphate (P_i) release by permeabilized fibres was recorded using the fluorescent protein MDCC-PBP, N-(2-[1-maleimidyl]ethyl)-7-diethylamino-coumarin-3 carboxamide phosphate binding protein. The steady-state ADP release rate was measured using a linked enzyme assay. The rates decreased five-fold during contraction in both intact and permeabilized fibres. In intact fibres the rate of heat + work output by actomyosin decreased from 134 ± s.e.m. 28 μW mg⁻¹ ( n = 17) at 0.055 s to 42% of this value at 0.25 s, and to 20% at 3.5 s. The force remained constant between 0.25 and 3.5 s. Similarly in permeabilized fibres the P_i release rate decreased from 5.00 ± 0.39 mmol l⁻¹ s⁻¹ at 0.055 s to 39% of this value at 0.25 s and to 19% at 0.5 s. The steady-state ADP release rate at 15 s was 21% of the P_i rate at 0.055 s. Using a single set of rate constants, the time courses of force, heat + work and P_i release were described by an actomyosin model that took account of the transition from the initial state (rest or rigor) to the contracting state, shortening and the consequent work against series elasticity, and reaction heats. The model suggests that increasing P_i concentration slows the cycle in intact fibres, and that changes in ATP and ADP slow the cycle in permeabilized fibres.     

The Smooth Muscle Myosin Seven Amino Acid Heavy Chain Insert's Kinetic Role in the Crossbridge Cycle for Mouse Bladder

The seven amino acid insert in the smooth muscle myosin heavy chain is thought to regulate the kinetics of contraction, contributing to the differences between fast and slow smooth muscle. The effects of this insert on force and stiffness were determined in bladder tissue of a transgenic mouse line expressing the insert SMB at one of three levels: an SMB wild type (+/+), an SMA homozygous type (−/−) and a heterozygous type (+/−). For skinned muscle, an increase in MgADP or inorganic phosphate (P_i) should shift the distribution of crossbridges in the actomyosin ATPase (AMATPase) to increase the relative population of the crossbridge state prior to ADP release and P_i release, respectively. Exogenous ADP increased force and stiffness in a manner consistent with increasing the Ca²⁺ concentration in both the +/+ and +/− mouse types. However, the −/− type showed a significantly greater increase in force than in stiffness suggesting that immediately prior to ADP release, the AMATPase either has an additional force producing isomerization state or a slower ADP dissociation rate for the −/− type compared to the +/+ or +/− types. Exogenous P_i led to a significantly greater decrease in stiffness than in force for all three mouse types suggesting that there is a force producing state prior to P_i release. In addition, the increase in P_i showed similar changes in the +/+ and −/− types whereas in the +/− type the decreases in both force and stiffness were greater than the other two mouse types indicating that the insert can affect the cooperativity between myosin heads. In conclusion, the seven amino acid insert modulates the kinetics and/or states of the AMATPase, which could lead to differences in the kinetics of contraction between fast and slow smooth muscle.     

Role of phosphate and calcium stores in muscle fatigue

Intensive activity of muscles causes a decline in performance, known as fatigue, that is thought to be caused by the effects of metabolic changes on either the contractile machinery or the activation processes. The concentration of inorganic phosphate (P_i) in the myoplasm ([P_i]_myo) increases substantially during fatigue and affects both the myofibrillar proteins and the activation processes. It is known that a failure of sarcoplasmic reticulum (SR) Ca²⁺ release contributes to fatigue and in this review we consider how raised [P_i]_myo contributes to this process. Initial evidence came from the observation that increasing [P_i]_myo causes reduced SR Ca²⁺ release in both skinned and intact fibres. In fatigued muscles the store of releasable Ca²⁺ in the SR declines mirroring the decline in SR Ca²⁺ release. In muscle fibres with inoperative creatine kinase the rise of [P_i]_myo is absent during fatigue and the failure of SR Ca²⁺ release is delayed. These results can all be explained if inorganic phosphate can move from the myoplasm into the SR during fatigue and cause precipitation of CaP_i within the SR. The relevance of this mechanism in different types of fatigue in humans is considered.     

Effect of ADP on slow-twitch muscle fibres of the rat: implications for muscle fatigue

Slow-twitch mechanically skinned fibres from rat soleus muscle were bathed in solutions mimicking the myoplasmic environment but containing different [ADP] (0.1 μ m to 1.0 m m ). The effect of ADP on sarcoplasmic reticulum (SR) Ca²⁺-content was determined from the magnitude of caffeine-induced force responses, while temporal changes in SR Ca²⁺-content allowed determination of the effective rates of the SR Ca²⁺-pump and of the SR Ca²⁺-leak. The SR Ca²⁺-pump rate, estimated at pCa (−log₁₀[Ca²⁺]) 7.8, was reduced by 20% as the [ADP] was increased from 0.1 to 40 μ m , with no further alteration when the [ADP] was increased to 1.0 m m . The SR Ca²⁺-leak rate constant was not altered by increasing [ADP] from 0.1 to 40 μ m , but was increased by 26% when the [ADP] was elevated to 1.0 m m . This ADP-induced SR Ca²⁺-leak was insensitive to ruthenium red but was abolished by 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ), indicating that the leak pathway is via the SR Ca²⁺-pump and not the SR Ca²⁺-release channel. The decrease in SR Ca²⁺-pump rate and SR Ca²⁺-leak rate when [ADP] was increased led to a 40% decrease in SR Ca²⁺-loading capacity. Elevation of [ADP] had only minor direct effects on the contractile apparatus of slow-twitch fibres. These results suggest that ADP has only limited depressing effects on the contractility of slow-twitch muscle fibres. This is in contrast to the marked effects of ADP on force responses in fast-twitch muscle fibres and may contribute to the fatigue-resistant nature of slow-twitch muscle fibres.     

Nitric oxide plays a role in the regulation of adrenal blood flow and adrenocorticomedullary functions in the llama fetus

The effect of temperature on isometric tension with and without the regulatory proteins tropomyosin and troponin was studied in bovine myocardium using a thin filament removal and reconstitution protocol. In control bovine myocardium, isometric tension increased linearly with temperature in the range 5–40 °C: isometric tension at 10 and 30 °C was 0.65 and 1.28 times that at 20 °C, respectively, with a Q ₁₀ of about 1.4. In actin filament-reconstituted myocardium without regulatory proteins, the temperature effect on isometric tension was less: isometric tension at 10 and 30 °C was 0.96 and 1.17 times that at 20 °C, respectively, with a Q ₁₀ of about 1.1. The temperature dependence of the apparent rate constants was studied using sinusoidal analysis. The temperature dependence of 2π b (rate constant of delayed tension phase) did not vary significantly with the regulatory proteins under the standard activating condition (5 m m MgATP, 8 m m P_i, 200 m m ionic strength, pCa 4.66, pH 7.00). Q ₁₀ for 2π b in control and actin filament-reconstituted myocardium was 3.8 and 4.0, respectively. There were two phases to the temperature dependence of 2π c (rate constant of quick recovery). In control and thin filament-reconstituted myocardium, Q ₁₀ for 2π c was approximately 5.5 in the low temperature range (≤ 25 °C) and 2.7 in the high temperature range (≥ 30 °C). In actin filament-reconstituted myocardium, Q ₁₀ for 2π c was 8.5 in the low temperature range and 3.6 in the high temperature range. The above results demonstrate that regulatory proteins augment the temperature dependence of isometric tension, indicating that the regulatory proteins may modify the actomyosin interaction.     

Limited oxygen diffusion accelerates fatigue development in mouse skeletal muscle

Isolated whole skeletal muscles fatigue more rapidly than isolated single muscle fibres. We have now employed this difference to study mechanisms of skeletal muscle fatigue. Isolated whole soleus and extensor digitorum longus (EDL) muscles were fatigued by repeated tetanic stimulation while measuring force production. Neither application of 10 m m lactic acid nor increasing the [K⁺] of the bath solution from 5 to 10 m m had any significant effect on the rate of force decline during fatigue induced by repeated brief tetani. Soleus muscles fatigued slightly faster during continuous tetanic stimulation in 10 m m [K⁺]. Inhibition of mitochondrial respiration with cyanide resulted in a faster fatigue development in both soleus and EDL muscles. Single soleus muscle fibres were fatigued by repeated tetani while measuring force and myoplasmic free [Ca²⁺] ([Ca²⁺]_i). Under control conditions, the single fibres were substantially more fatigue resistant than the whole soleus muscles; tetanic force at the end of a series of 100 tetani was reduced by about 10% and 50%, respectively. However, in the presence of cyanide, fatigue developed at a similar rate in whole muscles and single fibres, and tetanic force at the end of fatiguing stimulation was reduced by ∼80%. The force decrease in the presence of cyanide was associated with a ∼50% decrease in tetanic [Ca²⁺]_i, compared with an increase of ∼20% without cyanide. In conclusion, lactic acid or [K⁺] has little impact on fatigue induced by repeated tetani, whereas hypoxia speeds up fatigue development and this is mainly due to an impaired Ca²⁺ release from the sarcoplasmic reticulum.     

Reactive oxygen species reduce myofibrillar Ca2+ sensitivity in fatiguing mouse skeletal muscle at 37°C

The mechanisms of muscle fatigue were studied in small muscle bundles and single fibres isolated from the flexor digitorum brevis of the mouse. Fatigue caused by repeated isometric tetani was accelerated at body temperature (37°C) when compared to room temperature (22°C). The membrane-permeant reactive oxygen species (ROS) scavenger, Tiron (5 m m ), had no effect on the rate of fatigue at 22°C but slowed the rate of fatigue at 37°C to that observed at 22°C. Single fibres were microinjected with indo-1 to measure intracellular calcium. In the accelerated fatigue at 37°C the tetanic [Ca²⁺]_i did not change significantly and the decline of maximum Ca²⁺-activated force was similar to that observed at 22°C. The cause of the greater rate of fatigue at 37°C was a large fall in myofibrillar Ca²⁺ sensitivity. In the presence of Tiron, the large fall in Ca²⁺ sensitivity was abolished and the usual decline in tetanic [Ca²⁺]_i was observed. This study confirms the importance of ROS in fatigue at 37°C and shows that the mechanism of action of ROS is a decline in myofibrillar Ca²⁺ sensitivity.     

Calmodulin kinase modulates Ca2+ release in mouse skeletal muscle

Activation of the contractile machinery in skeletal muscle is initiated by the action-potential-induced release of Ca²⁺ from the sarcoplasmic reticulum (SR). Several proteins involved in SR Ca²⁺ release are affected by calmodulin kinase II (CaMKII)-induced phosphorylation in vitro , but the effect in the intact cell remains uncertain and is the focus of the present study. CaMKII inhibitory peptide or inactive control peptide was injected into single isolated fast-twitch fibres of mouse flexor digitorum brevis muscles, and the effect on free myoplasmic [Ca²⁺] ([Ca²⁺]_i) and force during different patterns of stimulation was measured. Injection of the inactive control peptide had no effect on any of the parameters measured. Conversely, injection of CaMKII inhibitory peptide decreased tetanic [Ca²⁺]_i by ≈25 %, but had no significant effect on the rate of SR Ca²⁺ uptake or the force-[Ca²⁺]_i relationship. Repeated tetanic stimulation resulted in increased tetanic [Ca²⁺]_i, and this increase was smaller after CaMKII inhibition. In conclusion, CaMKII-induced phosphorylation facilitates SR Ca²⁺ release in the basal state and during repeated contractions, providing a positive feedback between [Ca²⁺]_i and SR Ca²⁺ release.     

Contractile effects of the exchange of cardiac troponin for fast skeletal troponin in rabbit psoas single myofibrils

The effects of the removal of fast skeletal troponin C (fsTnC) and its replacement by cardiac troponin C (cTnC) and the exchange of fast skeletal troponin (fsTn) for cardiac troponin (cTn) were measured in rabbit fast skeletal myofibrils. Electrophoretic analysis of myofibril suspensions indicated that replacement of fsTnC or exchange of fsTn with cTnC or cTn was about 90% complete in the protocols used. Mechanical measurements in single myofibrils, which were maximally activated by fast solution switching, showed that replacement of fsTnC with cTnC reduced the isometric tension, the rate of tension rise following a step increase in Ca²⁺ ( k _act ), and the rate of tension redevelopment following a quick release and restretch ( k _tr ), but had no effect on the kinetics of the fall in tension when the concentration of inorganic phosphate (P_i) was abruptly increased ( k _Pi(+)). These data suggest that the chimeric protein produced by cTnC replacement in fsTn alters those steps controlling the weak-to-strong crossbridge attachment transition. Inefficient signalling within the chimeric troponin may cause these changes. However, replacement of fsTn by cTn had no effect on maximal isometric tension, k _act or k _tr , suggesting that these mechanics are largely determined by the isoform of the myosin molecule. Replacement of fsTn by cTn, on the other hand, shifted the pCa₅₀ of the pCa-tension relationship from 5.70 to 6.44 and reduced the Hill coefficient from 3.3 to 1.4, suggesting that regulatory protein isoforms primarily alter Ca²⁺ sensitivity and the cooperativity of the force-generating mechanism.     

Inorganic Phosphate Affects the pCa–Force Relationship More than the pCa–ATPase by Increasing the Rate of Dissociation of Force Generating Cross-Bridges in Skinned Fibers from Both EDL and Soleus Muscles of the Rat

The effect of inorganic phosphate (Pi) on Ca2+ -activation of actomyosin ATPase activity and force in permeabilized (skinned) single extensor digitorum longus (EDL) and soleus muscle fibers of the rat were investigated. Increasing concentrations of Pi decreased force more than ATPase rate at all Ca2+ concentrations and this effect was more pronounced at submaximal Ca2+ -activation. Increasing Pi caused both the normalized pCa-ATPase and pCa-force relationship to be shifted to a higher Ca2+ concentration. At all Ca2+ concentrations ATPase was activated at a lower concentration of Ca2+ than force and this difference in Ca2+ concentration required for the activation of ATPase and force was greater in fast-twitch (EDL) than in slow twitch (soleus) muscle. Soleus muscle pCa-ATPase and pCa-force curves were more sensitive to Ca2+ (pCa50 = 5.97 and 5.89, respectively) than EDL (pCa50 = 5.68 and 5.54, respectively). Finally the shape of the pCa-ATPase and pCa-force curves was similar and not affected by Pi. Analysis shows that Pi increases the rate of dissociation of force generating myosin cross-bridges (ratio of ATPase/force (g(app at all Ca2+ concentration, especially at submaximal Ca2+ -activation levels. Pi effects on g(app) are discussed in terms Pi interacting with the isomerization high force AM*ADP states to form high force transitional AM*ADP*Pi* states which facilitate the dissociation of ADP from AM*ADP. Increasing Ca2+ during Ca2+ -activation of the fibers is associated with a progressive decrease in rate of dissociation of force generating myosin cross-bridges g(app).     

Effects of Thyroxine on Myosin Isoform Expression and Mechanical Properties in Guinea-Pig Smooth Muscle

Information on the effects of thyroid hormone on smooth muscle contractile protein expression and mechanical properties is sparse. We have addressed the following questions. (1) Can thyroxine hormone alter myosin isoform composition in smooth muscle? (2) Can a change in myosin isoform composition lead to altered mechanical properties in smooth muscle? (3) Are alterations, if occurring, equal in fast and slow smooth muscle types? Guinea-pigs were treated with thyroxine (T₄) for 12 days. Control animals were given physiological saline solution. Maximal unloaded shortening velocity ( V _max) was measured in chemically skinned, maximally activated muscle preparations from the aorta and the taenia coli. V _max increased following thyroxine treatment, by approximately 20 % in the taenia coli. In the aorta, no significant increase in V _max could be detected. The sensitivity of isometric force to inorganic phosphate (P_i) was increased in the taenia coli following thyroxine treatment. The expression of mRNA (determined with RT-PCR) for the myosin heavy chain with the seven amino acid insert increased by approximately 70 % in the aorta and about 25 % in the taenia coli following thyroxine treatment. Western blot analysis showed an increase in the inserted myosin heavy chain form in the taenia coli. Expression of mRNA for the myosin essential light chains and the corresponding proteins did not change significantly in either muscle type. No alterations in non-muscle myosin heavy chain isoforms could be detected after thyroxine treatment. In conclusion, thyroxine treatment alters the isoform composition of myosin in fast and slow smooth muscles in vivo . This change is sufficient to increase shortening velocity and sensitivity of isometric force to P_i in the fast, but not in the slow, smooth muscle type.     

Phosphorylated guanidinoacetate partly compensates for the lack of phosphocreatine in skeletal muscle of mice lacking guanidinoacetate methyltransferase

The effects of creatine (Cr) absence in skeletal muscle caused by a deletion of guanidinoacetate methyltransferase (GAMT) were studied in a knockout mouse model by in vivo ³¹P magnetic resonance (MR) spectroscopy. ³¹P MR spectra of hindleg muscle of GAMT-deficient (GAMT–/–) mice showed no phosphocreatine (PCr) signal and instead showed the signal for phosphorylated guanidinoacetate (PGua), the immediate precursor of Cr, which is not normally present. Tissue pH did not differ between wild-type (WT) and GAMT–/–  mice, while relative inorganic phosphate (P_i) levels were increased in the latter. During ischaemia, PGua was metabolically active in GAMT–/–  mice and decreased at a rate comparable to the decrease of PCr in WT mice. However, the recovery rate of PGua in GAMT–/– mice after ischaemia was reduced compared to PCr in WT mice. Saturation transfer measurements revealed no detectable flux from PGua to γ-ATP, indicating severely reduced enzyme kinetics. Supplementation of Cr resulted in a rapid increase in PCr signal intensity until only this resonance was visible, along with a reduction in relative P_i values. However, the PGua recovery rate after ischaemia did not change. Our results show that despite the absence of Cr, GAMT–/– mice can cope with mild ischaemic stress by using PGua for high energy phosphoryl transfer. The reduced affinity of creatine kinase (CK) for (P)Gua only becomes apparent during recovery from ischaemia. It is argued that absence of Cr causes the higher relative P_i concentration also observed in animals lacking muscle CK, indicating an important role of the CK system in P_i homeostasis.     

High temperature does not alter fatigability in intact mouse skeletal muscle fibres

Intense activation of skeletal muscle results in fatigue development, which involves impaired function of the muscle cells resulting in weaker and slower contractions. Intense muscle activity also results in increased heat production and muscle temperature may rise by up to ∼6°C. Hyperthermia is associated with impaired exercise performance in vivo and recent studies have shown contractile dysfunction and premature fatigue development in easily fatigued muscle fibres stimulated at high temperatures and these defects were attributed to oxidative stress. Here we studied whether fatigue-resistant soleus fibres stimulated at increased temperature show premature fatigue development and whether increasing the level of oxidative stress accelerates fatigue development. Intact single fibres or small bundles of soleus fibres were fatigued by 600 ms tetani given at 2 s intervals at 37°C and 43°C, which is the highest temperature the muscle would experience in vivo . Tetanic force in the unfatigued state was not significantly different at the two temperatures. With 100 fatiguing tetani, force decreased by ∼15% at both temperatures; the free cytosolic [Ca²⁺] (assessed with indo-1) showed a similar ∼10% decrease at both temperatures. The oxidative stress during fatigue at 43°C was increased by application of 10 μ m hydrogen peroxide or tert-butyl hydroperoxide and this did not cause premature fatigue development. In summary, fatigue-resistant muscle fibres do not display impaired contractility and fatigue resistance at the highest temperature that mammals, including humans, would experience in vivo . Thus, intrinsic defects in fatigue-resistant muscle fibres cannot explain the decreased physical performance at high temperatures.     

The structural basis of the increase in isometric force production with temperature in frog skeletal muscle

X-ray diffraction patterns were recorded from isolated single fibres of frog skeletal muscle during isometric contraction at temperatures between 0 and 17°C. Isometric force was 43 ± 2% (mean ± s.e.m. , n = 10) higher at 17°C than 0°C. The intensity of the first actin layer line increased by 57 ± 18% ( n = 5), and the ratio of the intensities of the equatorial 1,1 and 1,0 reflections by 20 ± 7% ( n = 10), signalling radial or azimuthal motions of the myosin head domains. The M3 X-ray reflection from the axial repeat of the heads along the filaments was 27 ± 4% more intense at 17°C, suggesting that the heads became more perpendicular to the filaments. The ratio of the intensities of the higher and lower angle peaks of the M3 reflection ( R _M3) was 0.93 ± 0.02 ( n = 5) at 0°C and 0.77 ± 0.02 at 17°C. These peaks are due to interference between the two halves of each myosin filament, and the R _M3 decrease shows that heads move towards the midpoint of the myosin filament at the higher temperature. Calculations based on a crystallographic model of the heads indicated that the observed R _M3 change corresponds to tilting of their light-chain domains by 9 deg, producing an axial displacement of 1.4 nm, which is equal to that required to strain the actin and myosin filaments under the increased force. We conclude that the higher force generated by skeletal muscle at higher temperature can be accounted for by axial tilting of the myosin heads.     

Change in contractile properties of human muscle in relationship to the loss of power and slowing of relaxation seen with fatigue

Slow relaxation from an isometric contraction is characteristic of acutely fatigued muscle and is associated with a decrease in the maximum velocity of unloaded shortening ( V _max) and both these phenomena might be due to a decreased rate of cross bridge detachment. We have compared the change in relaxation rate with that of various parameters of the force–velocity relationship over the course of an ischaemic series of fatiguing contractions and subsequent recovery using the human adductor pollicis muscle working in vivo at approximately 37°C in nine healthy young subjects. Maximal isometric force ( F ₀) decreased from 91.0 ± 1.9 to 58.3 ± 3.5 N (mean ± s.e.m. ). Maximum power decreased from 53.6 ± 4.0 to 17.7 ± 1.2 (arbitrary units) while relaxation rate declined from −10.3 ± 0.38 to −2.56 ± 0.29 s⁻¹. V _max showed a smaller relative change from 673 ± 20 to 560 ± 46 deg s⁻¹ and with a time course that differed markedly from that of slowing of relaxation, showing very little change until late in the series of contractions. Curvature of the force–velocity relationship increased ( a/F ₀ decreasing from 0.22 ± 0.02 to 0.11 ± 0.02) with fatigue and with a time course that was similar to that of the loss of power and the slowing of relaxation. It is concluded that for human muscle working at a normal physiological temperature the change in curvature of the force–velocity relationship with fatigue is a major cause of loss of power and may share a common underlying mechanism with the slowing of relaxation from an isometric contraction.     

Neurokinin-1 receptor desensitization to consecutive microdialysis infusions of substance P in human skin

Unloaded shortening velocity ( V ₀) of human triceps surae muscle was measured in vivo by applying the 'slack test', originally developed for determining V ₀ of single muscle fibres, to voluntary contractions at varied activation levels (ALs). V ₀ was measured from 10 subjects at five different ALs defined as a fraction (5, 10, 20, 40 and 60%) of the maximum voluntary contraction (MVC) torque. Although individual variability was apparent, V ₀ tended to increase with AL  ( R ²= 0.089; P = 0.035)  up to 60%MVC (8.6 ± 2.6 rad s⁻¹). This value of V ₀ at 60%MVC was comparable to the maximum shortening velocity of plantar flexors reported in the previous studies. Electromyographic analysis showed that the activities of soleus, medial gastrocnemius and lateral gastrocnemius muscles increased with AL during isometric contraction and after the application of quick release in a similar manner. Also, it showed that the activity of an antagonist, tibialis anterior muscle, was negligible, even though a slight increase took place after the quick release of agonist. Correlation analysis showed that there were no significant correlations between V ₀ and MVC torque normalized with respect to body mass, although the correlation coefficient was relatively high at low ALs. The results suggest that in human muscle, V ₀ represents the unloaded velocity of the fastest muscle fibres recruited, and increases with AL possibly because of progressive recruitment of faster fibres. Individual variability may be explained, at least partially, by the difference in fibre-type composition.     

Clathrin-mediated endocytosis in snake motor terminals is directly facilitated by intracellular Ca2+

At the snake neuromuscular junction, low temperature (LT, 5–7°C) blocks clathrin-mediated endocytosis (CME) while exocytosis is largely unaffected. Thus compensatory endocytosis that normally follows transmitter release is inhibited, or 'delayed' until the preparation is warmed to room temperature (RT). This delay was exploited to observe how changes in bulk [Ca²⁺]_i directly affect CME. Motor terminals were loaded with fura-2 to monitor [Ca²⁺]_i. With brief stimulation at LT, [Ca²⁺]_i transiently increased but returned to baseline (∼63 n m ) in < 8 min. After 15 min at LT, [Ca²⁺]_i was altered by incubating preparations in the Ca²⁺ ionophore ionomyocin. Preparations were then warmed to RT to initiate delayed endocytosis, which was quantified as uptake of the fluorescent optical probe sulforhodamine 101. Endocytosis was more rapid when [Ca²⁺]_i increased; the rate at 300 n m Ca²⁺ was ∼double that under basal conditions. Thus the rate of CME – isolated from stimulation, transmitter release, and other forms of endocytosis – is directly influenced by intraterminal Ca²⁺.     

Temperature dependence of the force-generating process in single fibres from frog skeletal muscle

Generation of force and shortening in striated muscle is due to the cyclic interactions of the globular portion (the head) of the myosin molecule, extending from the thick filament, with the actin filament. The work produced in each interaction is due to a conformational change (the working stroke) driven by the hydrolysis of ATP on the catalytic site of the myosin head. However, the precise mechanism and the size of the force and length step generated in one interaction are still under question. Here we reinvestigate the endothermic nature of the force-generating process by precisely determining, in tetanised intact frog muscle fibres under sarcomere length control, the effect of temperature on both isometric force and force response to length changes. We show that raising the temperature: (1) increases the force and the strain of the myosin heads attached in the isometric contraction by the same amount (∼70 %, from 2 to 17 °C); (2) increases the rate of quick force recovery following small length steps (range between −3 and 2 nm (half-sarcomere)⁻¹) with a Q ₁₀ (between 2 and 12 °C) of 1.9 (releases) and 2.3 (stretches); (3) does not affect the maximum extent of filament sliding accounted for by the working stroke in the attached heads (10 nm (half-sarcomere)⁻¹). These results indicate that in isometric conditions the structural change leading to force generation in the attached myosin heads can be modulated by temperature at the expense of the structural change responsible for the working stroke that drives filament sliding. The energy stored in the elasticity of the attached myosin heads at the plateau of the isometric tetanus increases with temperature, but even at high temperature this energy is only a fraction of the mechanical energy released by attached heads during filament sliding.     

Location of C-protein,H-protein and X-protein in rabbit skeletal muscle fibre types

Summary The locations of C-protein, H-protein and X-protein in rabbit psoas, plantaris and soleus muscles have been investigated with fluorescently tagged specific antibodies. Two systems have been examined: isolated myofibrils allowed the locations of these proteins within the sarcomere to be determined, while cryosections allowed a comparison of the amounts of these proteins between different types of fibre in the three muscles.Using antibody-labelled cryosections, we find that the amounts of each of these proteins depends closely on the fibre type. In all the muscles studied, C-protein is present in the largest amounts in fast white and fast intermediate fibres and is absent from slow red fibres, while X-protein is absent from fast white fibres and is present in the largest amounts in fast and slow red fibres. In psoas muscle, H-protein is present in the largest amounts in fast white fibres and is absent in fast and slow red fibres. In plantaris muscle, however, H-protein is absent from fast white fibres but occurs in some slow red fibres.All psoas myofibrils label with anti-C and anti-H and a minority label with anti-X. In each case the pattern of labelling is a zone in each half of the A-band. Measured across the middle of the A-band, the zones for H-protein are much closer together than for C-protein; the centre-to-centre spacings are 0.35 µm for anti-H and 0.64 µm for anti-C. The fluorescent zones for X-protein are slightly but significantly closer (0.52 µm) than those for C-protein. All soleus myofibrils label with anti-X but the centre-to-centre spacing was greater (0.67 µm). With plantaris myofibrils, where labelling occurs with anti-C or anti-H, the spacings resemble those in psoas myofibrils, but with anti-X the spacing resembles that in soleus myofibrils.The spacing of the fluorescent zones in an A-band, whether produced by anti-C, anti-X or anti-H does not vary with sarcomere length. We conclude that X-protein and H-protein, like C-protein, are thick filament components.With both fibres and myofibrils, there is no simple relationship between the amount of X-protein and the amount of C-protein. Many fast intermediate fibres in psoas and plantaris muscle label as strongly with anti-C as do fast white fibres but also label as strongly with anti-X as do fast and slow red fibres. Similarly, some psoas and many plantaris myofibrils label strongly with both antibodies. We conclude that C-protein and X-protein can coexist on thick filaments but do not compete for the same sites.