The Active State of Mammalian Skeletal Muscle

A new technique is proposed for computing the active state of striated muscle, based on the three component model of Fenn and Marsh (8) and of Hill (7). The method permits calculation of the time course of the active state from its peak to the time at which maximum isometric twitch tension is reached. The intormation required for the calculation can be obtained from a single muscle without moving it from its mount in the lever system. The time course of the active state proved to be a function of the length of the muscle. This length dependency led to the predictions that (a) the length at which maximum force is developed during tetanic stimulation is different from that at which it is developed during a twitch, and (b) the tetanus-twitch tension ratio is a function of length. Both predictions were verified in a series of experiments on the rat gracilis anticus muscle at 17.5°C.     

Immunolocalization of Sarcoplasmic Reticulum Proteins in Mammalian Skeletal Muscle Fibers

SYNOPSIS. The sarcoplasmic reticulum is the intracellular membranesystem in skeletal muscle fibers which regulates the Ca^2$ concentrationof the myofibril and thereby the contraction relaxation cycle. In the past the proposed explanation for the differences inthe contractile properties of fast and slow skeletal fibershas been attributed mainly to quantitative rather than qualitativedifferences in the structure, function and molecular compositionof the sarcoplasmic reticulum of these two fiber types. Recentimmunocytochemical and biochemical studies have, however, clearlydemonstrated that the Ca^2$-ATPase of the sarcoplasmic reticulumin slow skeletal fibers is structurally and thus perhaps alsofunctionally related to that of the cardiac fibers, but distinctlydifferent from that of fast skeletal fibers. Furthermore similarstudies have shown that phospholamban, a cardiac sarcoplasmicreticulum protein believed to modulate the activity of the cardiacCa^2$-ATPase, is also present in slow but not fast skeletal fibers. The availability of antibodies specific to the fast and slowisoforms of the Ca^2$-ATPase, and to phospholamban will now enableus to apply immunocytochemical labeling techniques to examinethe effect of neuronal and other physiological signals on theregulation of the gene expression of sarcoplasmic reticulumproteins at the cellular level.     

Mechanism of Ion Permeation in Skeletal Muscle Chloride Channels

Voltage-gated Cl⁻ channels belonging to the ClC family exhibit unique properties of ion permeation and gating. We functionally probed the conduction pathway of a recombinant human skeletal muscle Cl⁻ channel (hClC-1) expressed both in Xenopus oocytes and in a mammalian cell line by investigating block by extracellular or intracellular I⁻ and related anions. Extracellular and intracellular I⁻ exert blocking actions on hClC-1 currents that are both concentration and voltage dependent. Similar actions were observed for a variety of other halide (Br⁻) and polyatomic (SCN⁻, NO₃ ⁻, CH₃SO₃ ⁻) anions. In addition, I⁻ block is accompanied by gating alterations that differ depending on which side of the membrane the blocker is applied. External I⁻ causes a shift in the voltage-dependent probability that channels exist in three definable kinetic states (fast deactivating, slow deactivating, nondeactivating), while internal I⁻ slows deactivation. These different effects on gating properties can be used to distinguish two functional ion binding sites within the hClC-1 pore. We determined K _D values for I⁻ block in three distinct kinetic states and found that binding of I⁻ to hClC-1 is modulated by the gating state of the channel. Furthermore, estimates of electrical distance for I⁻ binding suggest that conformational changes affecting the two ion binding sites occur during gating transitions. These results have implications for understanding mechanisms of ion selectivity in hClC-1, and for defining the intimate relationship between gating and permeation in ClC channels.     

Nerve Stump Length and Membrane Changes in Denervated Skeletal Muscle

THE effect of nerve stump length on the rate of failure of neuromuscular transmission and on the degeneration of the neuromuscular apparatus has been studied in some detail^1–4 and there is general agreement that neuromuscular transmission fails and the nerve terminals degenerate more rapidly if the nerve is cut close to the muscle than if it is cut far away.     

The Myosin Inhibitor Blebbistatin Stabilizes the Super-Relaxed State in Skeletal Muscle

The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX.     

The Myosin Inhibitor Blebbistatin Stabilizes the Super-Relaxed State in Skeletal Muscle

The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX.     

Membrane Currents in Mammalian Ventricular Heart Muscle Fibers Using a Voltage-Clamp Technique

Bundles of sheep ventricular fibers were voltage-clamped utilizing a modified sucrose gap technique and intracellular voltage control. An action potential was fired off in the usual way, and the clamp circuit was switched on at preselected times during activity. Clamping the membrane back to its resting potential during the early part of an action potential resulted in a surge of inward current. The initial amplitude of this current surge decreased as the clamp was switched on progressively later during the action potential. Inward current decreasing as a function of time was also recorded if the membrane potential was clamped beyond the presumed K equilibrium potential (to -130 mv). Clamping the membrane to the inside positive range (+40 mv to +60 mv) at different times of an action potential resulted in a step of outward current which was not time-dependent. The results suggest that normal repolarization of sheep ventricle depends on a time-dependent decrease of inward current (Na, Ca) rather than on a time-dependent increase of outward current (K).     

Membrane Conductances and Spectral Sensitivities of Pecten Photoreceptors

The electrical and spectral properties of depolarizing (proximal) and hyperpolarizing (distal) photoreceptors in the eye of the scallop, Pecten irradians, were examined. Both depolarizing and hyperpolarizing responses are associated with an increase in membrane conductance; in addition, the depolarizing response is characterized by a secondary decrease in conductance at light intensities which inactivate the response. Both responses can be reversed in polarity by applied current across the cell membrane. The depolarizing response has a reversal potential of approximately +10 mv, whereas the estimated reversal potential for the hyperpolarizing response is near -70 mv. The two responses have the same spectral sensitivity function, which agrees with a Dartnall nomogram for a rhodospin with a λ_max at 500 nm. It is suggested that the photochemical reactions produce different end products which give responses of opposite polarity in proximal and distal cells, or alternatively, that the reactions of the respective cell membranes to the same end product are different.     

Kinetic Analysis of the Inhibitory Effect of Glibenclamide on KATP Channels of Mammalian Skeletal Muscle

We investigated the block of K_ATP channels by glibenclamide in inside-out membrane patches of rat flexor digitorum brevis muscle. (1) We found that glibenclamide inhibited K_ATP channels with an apparent K _i of 63 nm and a Hill coefficient of 0.85. The inhibition of K_ATP channels by glibenclamide was unaffected by internal Mg²⁺. (2) Glibenclamide altered all kinetic parameters measured; mean open time and burst length were reduced, whereas mean closed time was increased. (3) By making the assumption that binding of glibenclamide to the sulphonylurea receptor (SUR) leads to channel closure, we have used the relation between mean open time, glibenclamide concentration and K _D to estimate binding and unbinding rate constants. We found an apparent rate constant for glibenclamide binding of 9.9 × 10⁷ m ⁻¹ sec⁻¹ and an unbinding rate of 6.26 sec⁻¹. (4) Glibenclamide is a lipophilic molecule and is likely to act on sulfonylurea receptors from within the hydrophobic phase of the cell membrane. The glibenclamide concentration within this phase will be greater than that in the aqueous solution and we have taken this into account to estimate a true binding rate constant of 1.66 × 10⁶ m ⁻¹ sec⁻¹. Received: 7 July 1996/Revised: 4 October 1996     

Monovalent Cationic Channel Activity in the Inner Membrane of Nuclei from Skeletal Muscle Fibers

Nuclear ion channels remain among the least studied and biophysically characterized channels. Although considerable progress has been made in characterizing calcium release channels in the nuclear membrane, very little is known regarding the properties of nuclear monovalent cationic channels. Here, we describe a method to isolate nuclei from adult skeletal muscle fibers that are suitable for electrophysiological experiments. Using this approach, we show for the first time, to our knowledge, that a nuclear monovalent cationic channel (NMCC) is prominently expressed in the inner membrane of nuclei isolated from flexor digitorum brevis skeletal muscle fibers of adult mice. In isotonic 140 mM KCl, the skeletal muscle NMCC exhibits a unitary conductance of ∼160 pS and high, voltage-independent open probability. Based on single-channel reversal potential measurements, NMCCs are slightly more permeable to potassium ions over sodium (P_K/P_Na = 2.68 ± 0.21) and cesium (P_K/P_Cs = 1.39 ± 0.03) ions. In addition, NMCCs do not permeate divalent cations, are inhibited by calcium ions, and demonstrate weak rectification in asymmetric Ca²⁺-containing solutions. Together, these studies characterize a voltage-independent NMCC in skeletal muscle, the properties of which are ideally suited to serve as a countercurrent mechanism during calcium release from the nuclear envelope.     

Effect of External and Internal pH Changes on K and Cl Conductances in the Muscle Fiber Membrane of a Giant Barnacle

The membrane potential and conductance of the giant muscle fiber of a barnacle (Balanus nubilus Darwin) were analyzed in relation to changes in the external (3.5–10.0) and the internal (4.7–9.6) pH, under various experimental conditions. A sharp increase in membrane conductance, associated with a large increase in conductance to Cl ions, was observed when the external pH was lowered to values below 5.0. The ratio of Cl to K conductance in normal barnacle saline is between –1/7 at pH 7.7, whereas at pH 4.0 the ratio is about 6–9. The behavior of the membrane in response to pH changes in a Cl-depleted muscle fiber shows that the K conductance decreases with decreasing external pH for the whole range of pH examined. A steep increase in Cl conductance is also observed when the internal pH of the fiber is lowered below 5.0. The K to Cl conductance ratio increases with increasing internal pH in a manner very similar to that found when the external pH is raised above 5.0. These facts suggest that the membrane is amphoteric with positive and negative fixed charge groups having dissociation constants such that at pH greater than 5, negative groups predominate and cations permeate more easily than anions, while at lower pH positive groups predominate, facilitating the passage of anions through the membrane.     

Skeletal Muscle Type Comparison of Subsarcolemmal Mitochondrial Membrane Phospholipid Fatty Acid Composition in Rat

The phospholipid composition of membranes can influence the physiological functioning of the cell or subcellular organelle. This association has been previously demonstrated in skeletal muscle, where cellular or subcellular membrane, specifically mitochondria, phospholipid composition is linked to muscle function. However, these observations are based on whole mixed skeletal muscle analysis, with little information on skeletal muscles of differing fiber-type compositions. These past approaches that used mixed muscle may have misidentified outcomes or masked differences. Thus, the purpose of this study was to compare the phospholipid fatty acid composition of subsarcolemmal (SS) mitochondria isolated from slow-twitch postural (soleus), fast-twitch highly oxidative glycolytic locomotory (red gastrocnemius), and fast-twitch oxidative glycolytic locomotory (plantaris) skeletal muscles. The main findings of the study demonstrated unique differences between SS mitochondrial membranes from postural soleus compared to the other locomotory skeletal muscles examined, specifically lower percentage mole fraction of phosphatidylcholine (PC) and significantly higher percentage mole fraction of saturated fatty acids (SFA) and lower n6 polyunsaturated fatty acids (PUFA), resulting in a lower unsaturation index. We also found that although there was no difference in the percentage mole fraction of cardiolipin (CL) between skeletal muscle types examined, CL of soleus mitochondrial membranes were approximately twofold more SFA and approximately two-thirds less PUFA, resulting in a 20–30% lower unsaturation and peroxidation indices. Thus, the results of this study indicate unique membrane lipid composition of mitochondria isolated from different skeletal muscle types, a potential consequence of their respective duty cycles.     

Leucine Supplementation Improves Skeletal Muscle Regeneration after Cryolesion in Rats

This study was undertaken in order to provide further insight into the role of leucine supplementation in the skeletal muscle regeneration process, focusing on myofiber size and strength recovery. Young (2-month-old) rats were subjected or not to leucine supplementation (1.35 g/kg per day) started 3 days prior to cryolesion. Then, soleus muscles were cryolesioned and continued receiving leucine supplementation until 1, 3 and 10 days later. Soleus muscles from leucine-supplemented animals displayed an increase in myofiber size and a reduction in collagen type III expression on post-cryolesion day 10. Leucine was also effective in reducing FOXO3a activation and ubiquitinated protein accumulation in muscles at post-cryolesion days 3 and 10. In addition, leucine supplementation minimized the cryolesion-induced decrease in tetanic strength and increase in fatigue in regenerating muscles at post-cryolesion day 10. These beneficial effects of leucine were not accompanied by activation of any elements of the phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin signalling pathway in the regenerating muscles. Our results show that leucine improves myofiber size gain and strength recovery in regenerating soleus muscles through attenuation of protein ubiquitination. In addition, leucine might have therapeutic effects for muscle recovery following injury and in some muscle diseases.     

Ion Current Rectification,Limiting and Overlimiting Conductances in Nanopores

Previous reports on Poisson-Nernst-Planck (PNP) simulations of solid-state nanopores have focused on steady state behaviour under simplified boundary conditions. These are Neumann boundary conditions for the voltage at the pore walls, and in some cases also Donnan equilibrium boundary conditions for concentrations and voltages at both entrances of the nanopore. In this paper, we report time-dependent and steady state PNP simulations under less restrictive boundary conditions, including Neumann boundary conditions applied throughout the membrane relatively far away from the nanopore. We simulated ion currents through cylindrical and conical nanopores with several surface charge configurations, studying the spatial and temporal dependence of the currents contributed by each ion species. This revealed that, due to slow co-diffusion of oppositely charged ions, steady state is generally not reached in simulations or in practice. Furthermore, it is shown that ion concentration polarization is responsible for the observed limiting conductances and ion current rectification in nanopores with asymmetric surface charges or shapes. Hence, after more than a decade of collective research attempting to understand the nature of ion current rectification in solid-state nanopores, a relatively intuitive model is retrieved. Moreover, we measured and simulated current-voltage characteristics of rectifying silicon nitride nanopores presenting overlimiting conductances. The similarity between measurement and simulation shows that overlimiting conductances can result from the increased conductance of the electric double-layer at the membrane surface at the depletion side due to voltage-induced polarization charges. The MATLAB source code of the simulation software is available via the website http://micr.vub.ac.be.     

The Potassium Flux Ratio in Skeletal Muscle As a Test for Independent Ion Movement

The flux ratio of potassium ions was measured on frog sartorius muscle under conditions in which a substantial net potassium loss occurs. Muscle fiber membrane potentials were measured under identical conditions. The observed flux ratios were compared with values calculated from a theoretical relation derived on the assumptions that the unidirectional fluxes are both passive and occur independently. The results favor the conclusion that the potassium fluxes across skeletal muscle membrane occur along passive electrochemical gradients and obey the independence principle.     

Isolation and Partial Characterization of an Acetylcholine Receptor-Enriched Membrane Fraction from Skeletal Muscle

Abstract

A procedure for purification of the bungarotoxin-binding fraction of sarcolemma from rabbit skeletal muscle is described. Muscle is homogenized in 0.25M sucrose without high salt extraction and membrane fractions separated initially by differential centrifugation procedures. An ultracentrifugation pellet enriched in cell surface and sarcoplasmic reticulum markers is further fractionated on a dextran gradient (density = 1.0 to 1.09). Two fractions are identified as sarcolemma according to high specific activities for lactoperoxidaseiodination, Na⁺, K⁺-ATPase and α-bungarotoxin-binding. No Ca⁺⁺, Mg⁺⁺-ATPase activity is found in these fractions. A third fraction, the dextran gradient pellet, is enriched in Ca⁺⁺, Mg⁺⁺-ATPase activity and lactoperoxidase iodinatable material and characterized by low bungarotoxin binding. This fraction represents a mixture of sarcoplasmic reticulum and transverse tubules with some sarcolemma contamination.     

Myostatin in the Pathophysiology of Skeletal Muscle

Myostatin is an endogenous, negative regulator of muscle growth determining both muscle fiber number and size. The myostatin pathway is conserved across diverse species ranging from zebrafish to humans. Experimental models of muscle growth and regeneration have implicated myostatin as an important mediator of catabolic pathways in muscle cells. Inhibition of this pathway has emerged as a promising therapy for muscle wasting. Here we discuss the recent developments and the controversies in myostatin research, focusing on the molecular and cellular mechanisms underlying the actions of myostatin on skeletal muscle and the potential therapeutic role of myostatin on muscle-related disorders.