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.     

Optical Imaging and Functional Characterization of the Transverse Tubular System of Mammalian Muscle Fibers using the Potentiometric Indicator di-8-ANEPPS

Potentiometric dyes are useful tools for studying membrane potential changes from compartments inaccessible to direct electrical recordings. In the past, we have combined electrophysiological and optical techniques to investigate, by using absorbance and fluorescence potentiometric dyes, the electrical properties of the transverse tubular system in amphibian skeletal muscle fibers. In this paper we expand on recent observations using the fluorescent potentiometric indicator di-8-ANEPPS to investigate structural and functional properties of the transverse tubular system in mammalian skeletal muscle fibers. Two-photon laser scanning confocal fluorescence images of live muscle fibers suggest that the distance between consecutive rows of transverse tubules flanking the Z-lines remains relatively constant in muscle fibers stretched to attain sarcomere lengths of up to 3.5 μm. Furthermore, the combined use of two-microelectrode electrophysiological techniques with microscopic fluorescence spectroscopy and imaging allowed us to compare the spectral properties of di-8-ANEPPS fluorescence in fibers at rest, with those of fluorescence transients recorded in stimulated fibers. We found that although the indicator has excitation and emission peaks at 470 and 588 nm, respectively, fluorescence transients display optimal fractional changes (13%/100 mV) when using filters to select excitation wavelengths in the 530–550 nm band and emissions beyond 590 nm. Under these conditions, results from tetanically stimulated fibers and from voltage-clamp experiments suggest strongly that, although the kinetics of di-8-ANEPPS transients in mammalian fibers are very rapid and approximate those of the surface membrane electrical recordings, they arise from the transverse tubular system membranes.     

Structure and Function of the Transverse Tubular System in Crustacean Muscle Fibers

The structure and function of the transverse tubular system(TTS) in two types of crustacean muscle fibers are examined.Giant fibers from the barnacle,Balanus nubilus, which are gradedlycontracting, are compared with allor-none twitch fibers fromthe crab, Carcinus maenas. Both fiber types were found to havedeep sarcolemmal invaginations which serve both to increasethe fiber surface area and to kfeep the length of the tubulesshort enough for electrotonic propagation.The ultrastructureof the tubular system in both types of fiber is compared.Thesystem is better developed in Carcinus than in Balanus, butthe slow Balanus fibers do have a relatively well developedTTS and sarcoplasmic reticulum in contrast to slow vertebratefibers. The apparent high, membrane-capacitance values of crustaceanfibers are the result of investigators not taking into considerationthe large increase in surface area due to the sarcolemmal infoldings.Thetubular membranes in Carcinus fibers were found to be permselectiveto chloride ions, and could be made to swell (as confirmed byelectron microscopy) by establishing an outward gradient forchloride across them. The capacitance of the tubular membranerelative to the plasma membrane was found to increase when thetubuleswere swollen. The implication of a fiber having two spatiallyseparated, differentially permeable membranes on excitation-contractioncoupling is discussed.     

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.     

Measurement of the Impedance of Frog Skeletal Muscle Fibers

Impedance measurements are necessary to determine the passive electrical properties of cells including the equivalent circuits of the several pathways for current flow. Such measurements are usually made with microelectrodes of high impedance (some 15 MΩ) over a wide frequency range (1-10,000 Hz) and so are subject to many errors. An input amplifier has been developed which has negligible phase shift in this frequency range because it uses negative feedback to keep tiny the voltage on top of the microelectrode. An important source of artifact is the extracellular potential produced by capacitive current flow through the wall of the microelectrodes and the effective resistance of the bathing solution. This artifact is reduced some 10 times by shielding the current microelectrode with a conductive paint. The residual artifact is analyzed, measured, and subtracted from our results. The interelectrode coupling capacitance is reduced below 2 × 10^-17 F and can be neglected. Phase and amplitude measurements are made with phase-sensitive detectors insensitive to noise. The entire apparatus is calibrated at different signal to noise ratios and the nature of the extracellular potential is investigated. The phase shift in the last 5-20 μm of the microelectrode tip is shown to be small and quite independent of frequency under several conditions. Experimental measurements of the phase characteristic of muscle fibers in normal Ringer are presented. The improvements in apparatus and the physiological significance of impedance measurements are discussed. It is suggested that the interpretation of impedance measurements is sensitive to small errors and so it is necessary to present objective evidence of the reliability of one's apparatus and measurements.     

Transverse Impedance of Single Frog Skeletal Muscle Fibers

The transverse electrical impedance of single frog skeletal muscle fibers was measured at 31 frequencies that ranged from 1 to 100,000 Hz. Each fiber was bathed entirely in Ringer's solution, but it was positioned so that a central length of 5 mm was in a hollow plastic disk and was electrically isolated from the ends of the fiber. The diameter of the segment of the fiber in the disk was measured and then the segment was pressed from opposite sides by two insulating wedges. Electrical current was passed transversely through the segment between two platinum-platinum black electrodes that were located in the pools of Ringer's solution within the disk. The results were corrected for stray parallel capacitance, series resistance of the Ringer's solution between the fiber and the electrodes, parallel shunt resistance around the fiber, and the phase shift of the measuring apparatus. A nonlinear least-squares routine was used to fit a lumped equivalent circuit to the data from six fibers. The equivalent circuit that was chosen for the fibers contained three parallel branches; each branch was composed of a resistor and a capacitor in series. The model also included a seventh adjustable parameter that was designed to account for the degree of compression of the fibers by the insulating wedges. The branches of the equivalent circuit were assumed to represent the electrical properties of: (a) the myoplasm in series with the membrane capacitance that was exposed directly to the pools of Ringer's solution; (b) the capacitance and series resistance of the transverse tubules that were exposed directly to the pools of Ringer's solution; (c) the membrane capacitance in series with the shunt resistance between the fibers and the insulating wedges. The results gave no indication that current entered the sarcoplasmic reticulum.     

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.     

Impedance of Frog Skeletal Muscle Fibers in Various Solutions

The linear circuit parameters of 140 muscle fibers in nine solutions are determined from phase measurements fitted with three circuit models: the disk model, in which the resistance to radial current flow is in the lumen of the tubules; the lumped model, in which the resistance is at the mouth of the tubules; and the hybrid model, in which it is in both places. The lumped model fails to fit the data. The disk and hybrid model fit the data, but the optimal circuit values of the hybrid model seem more reasonable. The circuit values depend on sarcomere length. The conductivity of the lumen of the tubules is less than, and varies in a nonlinear manner with, the conductivity of the bathing solution, suggesting that the tubules are partially occluded by some material like basement membrane which restricts the mobility of ions and has fixed charge. The x2.5 hypertonic sucrose solution used in many voltage clamp experiments produces a large increase in the radial resistance, suggesting that control of the potential across the tubular membranes would be difficult to achieve. Glycerol-treated fibers have 90% of their tubular system insulated from the extracellular solution and 10% connected to the extracellular solution through a high resistance. We discuss the implications of our results for calculations of the nonlinear properties of muscle fibers, including the action potential and the radial spread of contraction.     

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca²⁺ signaling is a fundamental physiological process in living cells. Ca²⁺ sparks are the elementary units of Ca²⁺ signaling in the striated muscle fibers that appear as highly localized Ca²⁺ release events mediated by ryanodine receptor (RyR) Ca²⁺ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca²⁺ sparks could provide information on the intracellular Ca²⁺ handling properties of healthy and diseased striated muscles. Although Ca²⁺ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.Detailed here is an experimental protocol for measuring Ca²⁺ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca²⁺ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca²⁺ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca²⁺ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP₃R) and RyR contributes to Ca²⁺ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca²⁺ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).     

A Theoretical Analysis of the Capacitance of Muscle Fibers Using a Distributed Model of the Tubular System

A model is developed to predict the changes in total capacitance (i.e. total charge stored divided by surface membrane potential) of the tubular system of muscle fibers. The tubular system is represented as a punctated disc and the area of membrane across which current flows is represented as a punctated annulus, the capacitance of the muscle fiber being proportional to this area. The area can be determined from a distributed model of the tubular system, in which the only resistance to radial current flow is presumed to be in the lumen of the tubules. Calculations are made of the variation of capacitance expected as the conductivity of the bathing solution is varied. These calculations include the effects of fixed charge in the tubular lumen and the effects of changes in the shape and volume of the tubular system in solutions of low conductivity. The calculated results fail to fit comparable experimental data, although they do qualitatively account for the known variation of the radial spread of contraction with conductivity of the bathing medium. It is pointed out that the existence of a significant "access resistance" at the mouth of the tubules might explain the discrepancy between theory and experiment.     

Caveolin-3 Is Associated with the T-Tubules of Mature Skeletal Muscle Fibers

Caveolae are abundant in skeletal muscle and their coat contains a specific isoform of caveolin, caveolin-3. It has been suggested that during muscle development, caveolin-3 is associated with the T-tubules, but that in adult muscle it is found on the plasma membrane only. We have studied the distribution of caveolin-3 in single skeletal muscle fibers from adult rat soleus by confocal immunofluorescence and by immunogold electron microscopy. We found that caveolin-3 occurs at the highest density on the plasma membrane but is also present in the core of the fibers, at the I-band/A-band interface where it is associated with the T-tubules. In neither domain of the muscle surface does caveolin-3 colocalize with the glucose transporter GLUT4 and there is no evidence for internalization of the caveolae in muscle.     

Silent Calcium Channels in Skeletal Muscle Fibers of the Crustacean Atya lanipes

The superficial (tonic) abdominal flexor muscles of Atya lanipes do not generate Ca²⁺ action potentials when depolarized and have no detectable inward Ca²⁺ current. These fibers, however, are strictly dependent on Ca²⁺ influx for contraction, suggesting that they depend on Ca²⁺-induced Ca²⁺ release for contractile activation. The nature of the communication between Ca²⁺ channels in the sarcolemmal/tubular membrane and Ca²⁺ release channels in the sarcoplasmic reticulum in this crustacean muscle was investigated. The effects of dihydropyridines on tension generation and the passive electrical response were examined in current-clamped fibers: Bay K 8644 enhanced tension about 100% but did not alter the passive electrical response; nifedipine inhibited tension by about 70%. Sr²⁺ and Ba²⁺ action potentials could be elicited in Ca²⁺-free solutions. The spikes generated by these divalent cations were abolished by nifedipine. As the Sr²⁺ or Ba²⁺ concentrations were increased, the amplitudes of the action potentials and their maximum rate of rise, V _max , increased and tended towards saturation. Three-microelectrode voltage-clamp experiments showed that even at high (138 mm) extracellular Ca²⁺ concentration the channels were silent, i.e., no inward Ca²⁺ current was detected. In Ca²⁺-free solutions, inward currents carried by 138 mm Sr²⁺ or Ba²⁺ were observed. The currents activated at voltages above −40 mV and peaked at about 0 mV. This voltage-activation profile and the sensitivity of the channels to dihydropyridines indicate that they resemble L-type Ca²⁺ channels. Peak inward current density values were low, ca.−33 μA/cm² for Sr²⁺ and −14 μA/cm² for Ba²⁺, suggesting that Ca²⁺ channels are present at a very low density. It is concluded that Ca²⁺-induced Ca²⁺ release in this crustacean muscle operates with an unusually high gain: Ca²⁺ influx through the silent Ca²⁺ channels is too low to generate a macroscopic inward current, but increases sufficiently the local concentration of Ca²⁺ in the immediate vicinity of the sarcoplasmic reticulum Ca²⁺ release channels to trigger the highly amplified release of Ca²⁺ required for tension generation. Received: 5 April 1999/Revised: 15 September 1999     

Endoplasmic Reticulum to Golgi Trafficking in Multinucleated Skeletal Muscle Fibers

The organization of membrane trafficking between endoplasmic reticulum and Golgi within multinucleated muscle fibers was analyzed. We found that markers for the compartment involved in endoplasmic reticulum to Golgi trafficking exhibited perinuclear as well as interfibrillar localization. Furthermore, these markers showed prominent colocalization with microtubules. To analyze membrane trafficking, we followed the temperature-controlled transport of the G protein of the mutant vesicular stomatitis virus, tsO45, in isolated myofibers. Perinuclear and cross-striated staining were seen at 39°C, while at 15°C a diffuse staining component appeared along a subset of interfibrillar microtubules. At 20°C, bright Golgi spots were seen to be associated with microtubules that appeared as circumnuclear rings and longitudinal bundles. Beneath the motor end plate, however, the organization of the Golgi elements and microtubules was found to be distinctive. Retrograde trafficking induced by brefeldin A resulted in the disappearance of the Golgi spots throughout the myofibers and the appearance of staining along microtubules. Thus, interfibrillar membranes seem to be active in protein export, and trafficking between endoplasmic reticulum and Golgi elements occurred throughout the myofibers. The results suggest that microtubules served as tracks for the two-way trafficking between the endoplasmic reticulum and the Golgi compartment.     

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.     

Trypanosoma cruzi: Histochemical Characterization of Parasitized Skeletal Muscle Fibers1

C57B1/6 mice were infected with Brasil strain Trypanosoma cruzi trypomastigotes. The leg muscles of the mice were serial-sectioned with a cryostat, and individual fibers were classified histochemically as type I or type II on the basis of succinic dehydrogenase or adenosine triphosphatase activity. Although markedly more type II fibers were present in the leg muscles, the percentage of infected type I fibers was nearly five-fold higher than type II. This is the first demonstration of a preferential in vivo distribution of T. cruzi in muscle fibers based upon muscle type.     

Voltage-Activated Elementary Calcium Release Events in Isolated Mouse Skeletal Muscle Fibers

The elementary Ca²⁺-release events underlying voltage-activated myoplasmic Ca²⁺ transients in mammalian muscle remain elusive. Here, we looked for such events in confocal line-scan (x,t) images of fluo-3 fluorescence taken from isolated adult mouse skeletal muscle fibers held under voltage-clamp conditions. In response to step depolarizations, spatially segregated fluorescence signals could be detected that were riding on a global increase in fluorescence. These discrete signals were separated using digital filtering in the spatial domain; mean values for their spatial half-width and amplitude were 1.99 ± 0.09 μm and 0.16 ± 0.005 ΔF/F ₀ (n = 151), respectively. Under control conditions, the duration of the events was limited by the pulse duration. In contrast, in the presence of maurocalcine, a scorpion toxin suspected to disrupt the process of repolarization-induced ryanodine receptor (RyR) closure, events uninterrupted by the end of the pulse were readily detected. Overall results establish these voltage-activated low-amplitude local Ca²⁺ signals as inherent components of the physiological Ca²⁺-release process of mammalian muscle and suggest that they result from the opening of either one RyR or a coherently operating group of RyRs, under the control of the plasma membrane polarization.     

Membrane Ion Conductances of Mammalian Skeletal Muscle in the Post-Decompression State after High-Pressure Treatment

Exposure of excitable tissues to hyperbaric environments has been shown to alter membrane ion conductances, but only little is known about the state of the membranes of intact cells in the post-decompression phase following a prolonged high-pressure treatment. Furthermore, almost nothing is known about high-pressure effects on skeletal muscle membranes. Therefore, we investigated changes to the input resistances, membrane potentials and voltage-gated membrane currents for sodium (INa), potassium (IK) and calcium (ICa) ions under voltage-clamp conditions in enzymatically isolated intact mammalian single fibers following a 3-hr high-pressure treatment up to 25 MPa at +4 degrees C. After a 3-hr 20 MPa treatment, the input resistance was increased but declined again for treatments with higher pressures. The resting membrane potentials were depolarized in the post-decompression phase following a 20-MPa high-pressure treatment; this could be explained by an increase in the Na+- over K+-permeability ratio and in intracellular [Na+]i. Following a 10-MPa high-pressure treatment, INa, IK and ICa amplitudes were similar compared to controls but were significantly reduced by 25 to 35% after a 3-hr 20-MPa high-pressure treatment. Interestingly, the voltage-dependent inactivation of INa and ICa seemed to be more stable at high pressures compared to the activation parameters, as no significant changes were found up to a 20-MPa treatment. For higher pressure applications (e.g., 25 MPa), there seemed to be a marked loss of membrane integrity and INa, IK and ICa almost disappeared.     

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).