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.     

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.     

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.     

Characterization of the cross-bridge force-generating step using inorganic phosphate and BDM in myofibrils from rabbit skeletal muscles

The inhibitory effects of inorganic phosphate (P_i) on isometric force in striated muscle suggest that in the ATPase reaction P_i release is coupled to force generation. Whether P_i release and the power stroke are synchronous events or force is generated by an isomerization of the quaternary complex of actomyosin and ATPase products (AM.ADP.P_i) prior to the following release of P_i is still controversial. Examination of the dependence of isometric force on [P_i] in rabbit fast (psoas; 5-15 °C) and slow (soleus; 15-20 °C) myofibrils was used to test the two-step hypothesis of force generation and P_i release. Hyperbolic fits of force-[P_i] relations obtained in fast and slow myofibrils at 15 °C produced an apparent asymptote as [P_i]∞ of 0.07 and 0.44 maximal isometric force (i.e. force in the absence of P_i) in psoas and soleus myofibrils, respectively, with an apparent K _d of 4.3 m m in both. In each muscle type, the force-[P_i] relation was independent of temperature. However, 2,3-butanedione 2-monoxime (BDM) decreased the apparent asymptote of force in both muscle types, as expected from its inhibition of the force-generating isomerization. These data lend strong support to models of cross-bridge action in which force is produced by an isomerization of the AM.ADP.P_i complex immediately preceding the P_i release step.     

Simultaneous recording of intramembrane charge movement components and calcium release in wild-type and S100A1−/− muscle fibres

In the preceding paper, we reported that flexor digitorum brevis (FDB) muscle fibres from S100A1 knock-out (KO) mice exhibit a selective suppression of the delayed, steeply voltage-dependent component of intra-membrane charge movement current termed Q _γ. Here, we use 50 μ m of the Ca²⁺ indicator fluo-4 in the whole cell patch clamp pipette, in addition to 20 m m EGTA and other constituents included for the charge movement studies, and calculate the SR Ca²⁺ release flux from the fluo-4 signals during voltage clamp depolarizations. Ca²⁺ release flux is decreased in amplitude by the same fraction at all voltages in fibres from S100A1 KO mice compared to fibres from wild-type (WT) littermates, but unchanged in time course at each pulse membrane potential. There is a strong correlation between the time course and magnitude of release flux and the development of Q _γ. The decreased Ca²⁺ release in KO fibres is likely to account for the suppression of Q _γ in these fibres. Consistent with this interpretation, 4-chloro- m -cresol (4–CMC; 100 μ m ) increases the rate of Ca²⁺ release and restores Q _γ at intermediate depolarizations in fibres from KO mice, but does not increase Ca²⁺ release or restore Q _γ at large depolarizations. Our findings are consistent with similar activation kinetics for SR Ca²⁺ channels in both WT and KO fibres, but decreased Ca²⁺ release in the KO fibres possibly due to shorter SR channel open times. The decreased Ca²⁺ release at each voltage is insufficient to activate Q _γ in fibres lacking S100A1.     

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.     

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.     

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.     

Metabolic Regulation of Ca2+ Release in Permeabilized Mammalian Skeletal Muscle Fibres

In the present study, the link between cellular metabolism and Ca²⁺ signalling was investigated in permeabilized mammalian skeletal muscle. Spontaneous events of Ca²⁺ release from the sarcoplasmic reticulum were detected with fluo-3 and confocal scanning microscopy. Mitochondrial functions were monitored by measuring local changes in mitochondrial membrane potential (with the potential-sensitive dye tetramethylrhodamine ethyl ester) and in mitochondrial [Ca²⁺] (with the Ca²⁺ indicator mag-rhod-2). Digital fluorescence imaging microscopy was used to quantify changes in the mitochondrial autofluorescence of NAD(P)H. When fibres were immersed in a solution without mitochondrial substrates, Ca²⁺ release events were readily observed. The addition of l -glutamate or pyruvate reversibly decreased the frequency of Ca²⁺ release events and increased mitochondrial membrane potential and NAD(P)H production. Application of various mitochondrial inhibitors led to the loss of mitochondrial [Ca²⁺] and promoted spontaneous Ca²⁺ release from the sarcoplasmic reticulum. In many cases, the increase in the frequency of Ca²⁺ release events was not accompanied by a rise in global [Ca²⁺]_i. Our results suggest that mitochondria exert a negative control over Ca²⁺ signalling in skeletal muscle by buffering Ca²⁺ near Ca²⁺ release channels.     

Interpolated twitches in fatiguing single mouse muscle fibres: implications for the assessment of central fatigue

An electrically evoked twitch during a maximal voluntary contraction (twitch interpolation) is frequently used to assess central fatigue. In this study we used intact single muscle fibres to determine if intramuscular mechanisms could affect the force increase with the twitch interpolation technique. Intact single fibres from flexor digitorum brevis of NMRI mice were dissected and mounted in a chamber equipped with a force transducer. Free myoplasmic [Ca²⁺] ([Ca²⁺]_i) was measured with the fluorescent Ca²⁺ indicator indo-1. Seven fibres were fatigued with repeated 70 Hz tetani until 40% initial force with an interpolated pulse evoked every fifth tetanus. Results showed that the force generated by the interpolated twitch increased throughout fatigue, being 9 ± 1% of tetanic force at the start and 19 ± 1% at the end ( P < 0.001). This was not due to a larger increase in [Ca²⁺]_i induced by the interpolated twitch during fatigue but rather to the fact that the force–[Ca²⁺]_i relationship is sigmoidal and fibres entered a steeper part of the relationship during fatigue. In another set of experiments, we observed that repeated tetani evoked at 150 Hz resulted in more rapid fatigue development than at 70 Hz and there was a decrease in force ('sag') during contractions, which was not observed at 70 Hz. In conclusion, the extent of central fatigue is difficult to assess and it may be overestimated when using the twitch interpolation technique.     

Differential effects of maurocalcine on Ca2+ release events and depolarization-induced Ca2+ release in rat skeletal muscle

Maurocalcine (MCa), a 33 amino acid toxin obtained from scorpion venom, has been shown to interact with the isolated skeletal-type ryanodine receptor (RyR1) and to strongly modify its calcium channel gating. In this study, we explored the effects of MCa on RyR1 in situ to establish whether the functional interaction of RyR1 with the voltage-sensing dihydropyridine receptor (DHPR) would modify the ability of MCa to interact with RyR1. In developing skeletal muscle cells the addition of MCa into the external medium induced a calcium transient resulting from RyR1 activation and strongly inhibited the effect of the RyR1 agonist chloro- m -cresol. In contrast, MCa failed to affect the depolarization-induced Ca²⁺ release. In intact adult fibres MCa did not induce any change in the cytosolic Ca²⁺ concentration. However, when the surface membrane was permeabilized and calcium release events were readily observable, MCa had a time-dependent dual effect: it first increased event frequency, from 0.060 ± 0.002 to 0.150 ± 0.007 sarcomere⁻¹ s⁻¹, and reduced the amplitude of individual events without modifying their spatial distribution. Later on it induced the appearance of long-lasting events resembling the embers observed in control conditions but having a substantially longer duration. We propose that the functional coupling of DHPRs and RyR1s within a Ca²⁺ release unit prevents MCa from either reaching its binding site or from being able to modify the gating not only of the RyR1s physically coupled to DHPRs but all RyR1s within the Ca²⁺ release unit.     

Mini-dystrophin restores L-type calcium currents in skeletal muscle of transgenic mdx mice

L-type calcium currents ( i _Ca) were recorded using the two-microelectrode voltage-clamp technique in single short toe muscle fibres of three different mouse strains: (i) C57/SV129 wild-type mice (wt); (ii) mdx mice (an animal model for Duchenne muscular dystrophy; and (iii) transgenically engineered mini-dystrophin (MinD)-expressing mdx mice. The activation and inactivation properties of i _Ca were examined in 2- to 18-month-old animals. Ca²⁺ current densities at 0 mV in mdx fibres increased with age, but were always significantly smaller compared to age-matched wild-type fibres. Time-to-peak (TTP) of i _Ca was prolonged in mdx fibres compared to wt fibres. MinD fibres always showed similar TTP and current amplitudes compared to age-matched wt fibres. In all three genotypes, the voltage-dependent inactivation and deactivation of i _Ca were similar. Intracellular resting calcium concentration ([Ca²⁺]_i) and the distribution of dihydropyridine binding sites were also not different in young animals of all three genotypes, whereas i _Ca was markedly reduced in mdx fibres. We conclude, that dystrophin influences L-type Ca²⁺ channels via a direct or indirect linkage which may be disrupted in mdx mice and may be crucial for proper excitation–contraction coupling initiating Ca²⁺ release from the sarcoplasmic reticulum. This linkage seems to be fully restored in the presence of mini-dystrophin.     

Changes in contractile activation characteristics of rat fast and slow skeletal muscle fibres during regeneration

Damaged skeletal muscle fibres are replaced with new contractile units via muscle regeneration. Regenerating muscle fibres synthesize functionally distinct isoforms of contractile and regulatory proteins but little is known of their functional properties during the regeneration process. An advantage of utilizing single muscle fibre preparations is that assessment of their function is based on the overall characteristics of the contractile apparatus and regulatory system and as such, these preparations are sensitive in revealing not only coarse, but also subtle functional differences between muscle fibres. We examined the Ca²⁺- and Sr²⁺-activated contractile characteristics of permeabilized fibres from rat fast-twitch (extensor digitorum longus) and slow-twitch (soleus) muscles at 7, 14 and 21 days following myotoxic injury, to test the hypothesis that fibres from regenerating fast and slow muscles have different functional characteristics to fibres from uninjured muscles. Regenerating muscle fibres had ∼10% of the maximal force producing capacity ( P _o) of control (uninjured) fibres, and an altered sensitivity to Ca²⁺ and Sr²⁺ at 7 days post-injury. Increased force production and a shift in Ca²⁺ sensitivity consistent with fibre maturation were observed during regeneration such that P _o was restored to 36–45% of that in control fibres by 21 days, and sensitivity to Ca²⁺ and Sr²⁺ was similar to that of control (uninjured) fibres. The findings support the hypothesis that regenerating muscle fibres have different contractile activation characteristics compared with mature fibres, and that they adopt properties of mature fast- or slow-twitch muscle fibres in a progressive manner as the regeneration process is completed.     

Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic β-cells

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic β-cells by a combination of electrophysiological recordings, intracellular Ca²⁺ ([Ca²⁺]_i) microfluorimetry and insulin release measurements. Addition of palmitate (1 m m , bound to fatty acid-free albumin) to intact islets exposed to 15 m m glucose increased the [Ca²⁺]_i by ∼30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca²⁺]_i and insulin release in the presence of tolbutamide and in islets depolarized by high K⁺ in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K⁺ channels (K_ATP channels). Palmitate (0.5 m m ) augmented exocytosis (measured as an increase in cell capacitance) in single β-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca²⁺ currents rose by ∼25% following addition of 0.5 m m palmitate, an effect that was abolished in the presence of 10 μ m isradipine indicating that the free fatty acid specifically acts on L-type Ca²⁺ channels. The actions of palmitate on exocytosis and Ca²⁺ currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K_ATP channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca²⁺ currents and an increased size of the RRP.     

Elementary properties of CaV1.3 Ca2+ channels expressed in mouse cochlear inner hair cells

Mammalian cochlear inner hair cells (IHCs) are specialized to process developmental signals during immature stages and sound stimuli in adult animals. These signals are conveyed onto auditory afferent nerve fibres. Neurotransmitter release at IHC ribbon synapses is controlled by L-type Ca_V1.3 Ca²⁺ channels, the biophysics of which are still unknown in native mammalian cells. We have investigated the localization and elementary properties of Ca²⁺ channels in immature mouse IHCs under near-physiological recording conditions. Ca_V1.3 Ca²⁺ channels at the cell pre-synaptic site co-localize with about half of the total number of ribbons present in immature IHCs. These channels activated at about −70 mV, showed a relatively short first latency and weak inactivation, which would allow IHCs to generate and accurately encode spontaneous Ca²⁺ action potential activity characteristic of these immature cells. The Ca_V1.3 Ca²⁺ channels showed a very low open probability (about 0.15 at −20 mV: near the peak of an action potential). Comparison of elementary and macroscopic Ca²⁺ currents indicated that very few Ca²⁺ channels are associated with each docked vesicle at IHC ribbon synapses. Finally, we found that the open probability of Ca²⁺ channels, but not their opening time, was voltage dependent. This finding provides a possible correlation between presynaptic Ca²⁺ channel properties and the characteristic frequency/amplitude of EPSCs in auditory afferent fibres.     

Defective regulation of contractile function in muscle fibres carrying an E41K β-tropomyosin mutation

A novel E41K β-tropomyosin (β-Tm) mutation, associated with congenital myopathy and muscle weakness, was recently identified in a woman and her daughter. In both patients, muscle weakness was coupled with muscle fibre atrophy. It remains unknown, however, whether the E41K β-Tm mutation directly affects regulation of muscle contraction, contributing to the muscle weakness. To address this question, we studied a broad range of contractile characteristics in skinned muscle fibres from the two patients and eight healthy controls. Results showed decreases (i) in speed of contraction at saturated Ca²⁺ concentration (apparent rate constant of force redevelopment ( k _tr) and unloaded shortening speed ( V ₀)); and (ii) in contraction sensitivity to Ca²⁺ concentration, in fibres from patients compared with controls, suggesting that the mutation has a negative effect on contractile function, contributing to the muscle weakness. To investigate whether these negative impacts are reversible, we exposed skinned muscle fibres to the Ca²⁺ sensitizer EMD 57033. In fibres from patients, 30 μ m of EMD 57033 (i) had no effect on speed of contraction ( k _tr and V ₀) at saturated Ca²⁺ concentration but (ii) increased Ca²⁺ sensitivity of contraction, suggesting a potential therapeutic approach in patients carrying the E41K β-Tm mutation.     

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.     

The Qγ component of intra-membrane charge movement is present in mammalian muscle fibres, but suppressed in the absence of S100A1

S100A1 is a Ca²⁺ binding protein that modulates excitation–contraction (EC) coupling in skeletal and cardiac muscle. S100A1 competes with calmodulin for binding to the skeletal muscle SR Ca²⁺ release channel (the ryanodine receptor type 1, RyR1) at a site that also interacts with the C-terminal tail of the voltage sensor of EC coupling, the dihydropyridine receptor. Ablation of S100A1 leads to delayed and decreased action potential evoked Ca²⁺ transients, possibly linked to altered voltage sensor activation. Here we investigate the effects of S100A1 on voltage sensor activation in skeletal muscle utilizing whole-cell patch clamp electrophysiology to record intra-membrane charge movement currents in isolated flexor digitorum brevis (FDB) muscle fibres from wild-type and S100A1 knock-out (KO) mice. In contrast to recent reports, we found that FDB fibres exhibit two distinct components of intra-membrane charge movement, an initial rapid component ( Q _β), and a delayed, steeply voltage dependent 'hump' component ( Q _γ) previously recorded primarily in amphibian but not mammalian fibres. Surprisingly, we found that Q _γ was selectively suppressed in S100A1 KO fibres, while the Q _β component of charge movement was unaffected. This result was specific to S100A1 and not a compensatory result of genetic manipulation, as transient intracellular application of S100A1 restored Q _γ. Furthermore, we found that exposure to the RyR1 inhibitor dantrolene suppressed a similar component of charge movement in FDB fibres. These results shed light on voltage sensor activation in mammalian muscle, and support S100A1 as a positive regulator of the voltage sensor and Ca²⁺ release channel in skeletal muscle EC coupling.     

Effects of Mg2+ and SR luminal Ca2+ on caffeine-induced Ca2+ release in skeletal muscle from humans susceptible to malignant hyperthermia

Regulation of the ryanodine receptor (RYR) by Mg²⁺ and SR luminal Ca²⁺ was studied in mechanically skinned malignant hyperthermia susceptible (MHS) and non-susceptible (MHN) fibres from human vastus medialis. Preparations were perfused with solutions mimicking the intracellular milieu and changes in [Ca²⁺] were detected using fura-2 fluorescence. At 1 m m cytosolic Mg²⁺, MHS fibres had a higher sensitivity to caffeine (2-40 m m ) than MHN fibres. The inhibitory effect of Mg²⁺ on caffeine-induced Ca²⁺ release was studied by increasing [Mg²⁺] of the solution containing 40 m m caffeine. Increasing [Mg²⁺] from 1 to 3 m m reduced the amplitude of the caffeine-induced Ca²⁺ transient by 77 ± 7.4 % ( n = 8) in MHN fibres. However, the caffeine-induced Ca²⁺ transient decreased by only 24 ± 8.1 % ( n = 9) in MHS fibres. In MHN fibres, reducing the Ca²⁺ loading period from 4 to 1 min (at 1 m m Mg²⁺) decreased the fraction of the total sarcoplasmic reticulum (SR) Ca²⁺ content released in response to 40 m m caffeine by 90.4 ± 6.2 % ( n = 6). However, in MHS fibres the response was reduced by only 31.2 ± 17.4 % ( n = 6) under similar conditions. These results suggest that human malignant hyperthermia (MH) is associated with reduced inhibition of the RYR by (i) cytosolic Mg²⁺ and (ii) SR Ca²⁺ depletion. Both of these effects may contribute to increased sensitivity of the RYR to caffeine and volatile anaesthetics.     

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.