Quantitative elemental x-ray imaging of timed calcium displacement from stores (JSR) in isolated single frog skeletal muscle fibers: Its efficacy in quantitation

We are performing a continuing series of experiments to describe the time course of fast physiological events in terms of morphology and microtopochemistry, using electron probe x-ray microanalysis in both the static probe (STP) and quantitative digital imaging (QDI) modes. As a model, we are using timed spatial displacements of elements (e.g. the release of calcium from JSR) during the process of excitation-contraction coupling in single, intact skeletal muscle fibers quick-frozen at known time intervals following electrical stimulation. There is considerable variance in the total calcium concentration ([Ca]t) among JSRs, which increases the requirement for widespread sampling to increase statistical confidence. Even at a low number of pixels/raster chosen for time economy, QDI seems well suited to deal with this variance because it covers a large number of JSRs in a reasonably short scanning time (64x64 pixels: ∽3 h; 128×128 pixels: ∽9 h). Here, we report on the efficacy of QDI in our experiments and compare the results with those obtained from STP.

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Quantitative x-ray elemental imaging in stimulated single intact skeletal muscle fibers: The fate of calcium in the presence of ryanodine

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102535 ◽

1988 ◽

Vol 46 ◽

pp. 90-91

Author(s):

J. Sommer ◽

P. Ingram ◽

A. LeFurgey ◽

R. Nassar ◽

T. High

Keyword(s):

Skeletal Muscle ◽

Electrical Stimulation ◽

Time Course ◽

Imaging System ◽

Quantitative Imaging ◽

Muscle Fibers ◽

Frog Skeletal Muscle ◽

X Ray ◽

Skeletal Muscle Fibers ◽

Freeze Dried

We are involved in a continuing series of experiments aimed at a complete description,in terms of morphology and quantitative topochemistry, of the time course of spatial distributions of physiologically important elements during excitation-contraction coupling (ECC) at different time intervals (fractions of msec) following electrical stimulation of single, intact frog skeletal muscle fibers. In this present study wg report such distributions for Ca after 1,2 and 3 min of electrical stimulation in the presence of 2x10-4 M ryanodine, an alkaloid that, in time, causes irreversible muscle contractures.Single, intact frog skeletal muscle fibers were quick-frozen, cryosectioned, freeze-substituted and in one case freeze-fractured. The freeze-dried cryosections were subjected to electron probe X-ray microanalysis (EPXMA) in a JEOL 1200EX analytical electron microscope equipped with a Tracor Northern X-ray detector and a fully quantitative imaging system. Both, 64/64 pixel images (ambient temp.), and small raster probes (cold stage,-115 °C) for better statistics, were obtained, each from the same section.

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Impairment of Ca2+ release in single Xenopusmuscle fibers fatigued at varied extracellular P O 2

Journal of Applied Physiology ◽

10.1152/jappl.2000.88.5.1743 ◽

2000 ◽

Vol 88 (5) ◽

pp. 1743-1748 ◽

Cited By ~ 21

Author(s):

Creed M. Stary ◽

Michael C. Hogan

Keyword(s):

Skeletal Muscle ◽

Muscle Fibers ◽

Rapid Onset ◽

Skeletal Muscle Fibers ◽

Significant Difference ◽

Force Recovery ◽

Series Of Experiments ◽

Initial Peak ◽

Time Point ◽

Text Condition

We tested the hypothesis that the mechanisms involved in the more rapid onset of fatigue when O2 availability is reduced in contracting skeletal muscle are similar to those when O2 availability is more sufficient. Two series of experiments were performed in isolated, single skeletal muscle fibers from Xenopus laevis. First, relative force and free cytosolic Ca2+concentrations ([Ca2+]c) were measured simultaneously in single fibers ( n = 6) stimulated at increasing frequencies (0.25, 0.33, 0.5, and 1 Hz) at an extracellular[Formula: see text] of either 22 or 159 Torr. Muscle fatigue (force = 50% of initial peak tension) occurred significantly sooner ( P < 0.05) during the low- (237 ± 40 s) vs. high-[Formula: see text]treatments (280 ± 38 s). Relative [Ca2+]c was significantly decreased from maximal values at the fatigue time point during both the high- (72 ± 4%) and low-[Formula: see text] conditions (78 ± 4%), but no significant difference was observed between the treatments. In the second series of experiments, using the same stimulation regime as the first, fibers ( n = 6) exposed to 5 mM caffeine immediately after fatigue demonstrated an immediate but incomplete relative force recovery during both the low- (89 ± 4%) and high-[Formula: see text] treatments (82 ± 3%), with no significant difference between treatments. Additionally, there was no significant difference in relative [Ca2+]c between the high- (100 ± 12% of prefatigue values) and low-[Formula: see text] treatments (108 ± 12%) on application of caffeine. These results suggest that in isolated, single skeletal muscle fibers, the earlier onset of fatigue that occurred during the low-extracellular[Formula: see text] condition was modulated through similar pathways as the fatigue process during the high and involved a decrease in relative peak [Ca2+]c.

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Ultraslow contractile inactivation in frog skeletal muscle fibers.

The Journal of General Physiology ◽

10.1085/jgp.96.1.47 ◽

1990 ◽

Vol 96 (1) ◽

pp. 47-56 ◽

Cited By ~ 3

Author(s):

C Caputo ◽

P Bolaños

Keyword(s):

Skeletal Muscle ◽

Time Course ◽

Muscle Fibers ◽

Membrane Depolarization ◽

Frog Muscle ◽

Slow Time ◽

Inactivation Curve ◽

Lanthanum Ions ◽

Skeletal Muscle Fibers ◽

Spontaneous Relaxation

After a contracture response, skeletal muscle fibers enter into a state of contractile refractoriness or inactivation. Contractile inactivation starts soon after membrane depolarization, and causes spontaneous relaxation from the contracture response. Here we demonstrate that contractile inactivation continues to develop for tens of seconds if the membrane remains in a depolarized state. We have studied this phenomenon using short (1.5 mm) frog muscle fibers dissected from the Lumbricalis brevis muscles of the frog, with a two-microelectrode voltage-clamp technique. After a contracture caused by membrane depolarization to 0 mV, from a holding potential of -100 mV, a second contracture can be developed only if the membrane is repolarized beyond a determined potential value for a certain period of time. We have used a repriming protocol of 1 or 2 s at -100 mV. After this repriming period a fiber, if depolarized again to 0 mV, may develop a second contracture, whose magnitude and time course will depend on the duration of the period during which the fiber was maintained at 0 mV before the repriming process. With this procedure it is possible to demonstrate that the inactivation process builds up with a very slow time course, with a half time of approximately 35 s and completion in greater than 100 s. After prolonged depolarizations (greater than 100 s), the repriming time course is slower and the inactivation curve (obtained by plotting the extent of repriming against the repriming membrane potential) is shifted toward more negative potentials by greater than 30 mV when compared with similar curves obtained after shorter depolarizing periods (10-30 s). These results indicate that important changes occur in the physical state of the molecular moiety that is responsible for the inactivation phenomenon. The shift of the inactivation curve can be partially reversed by a low concentration (50 microM) of lanthanum ions. In the presence of 0.5 mM caffeine, larger responses can be obtained even after prolonged depolarization periods, indicating that the fibers maintain their capacity to liberate calcium.

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Steady State Relation between Cytoplasmic Free Ca2+ Concentration and Force in Intact Frog Skeletal Muscle Fibers

The Journal of General Physiology ◽

10.1085/jgp.111.4.505 ◽

1998 ◽

Vol 111 (4) ◽

pp. 505-519 ◽

Cited By ~ 11

Author(s):

Masato Konishi ◽

Masaru Watanabe

Keyword(s):

Skeletal Muscle ◽

Steady State ◽

Isometric Force ◽

Muscle Fibers ◽

Skinned Fibers ◽

Hill Coefficient ◽

Experimental Conditions ◽

Skeletal Muscle Fibers ◽

Force Relation ◽

Series Of Experiments

The steady state relation between cytoplasmic Ca2+ concentration ([Ca2+]i) and force was studied in intact skeletal muscle fibers of frogs. Intact twitch fibers were injected with the dextran-conjugated Ca2+ indicator, fura dextran, and the fluorescence signals of fura dextran were converted to [Ca2+]i using calibration parameters previously estimated in permeabilized muscle fibers (Konishi and Watanabe. 1995. J. Gen. Physiol. 106:1123–1150). In the first series of experiments, [Ca2+]i and isometric force were simultaneously measured during high K+ depolarization. Slow changes in [Ca2+]i and force induced by 15–30 mM K+ appeared to be in equilibrium, as instantaneous [Ca2+]i versus force plot tracked the common path in the rising and relaxation phases of K+ contractures. In the second series of experiments, 2,5-di-tert-butylhydroquinone (TBQ), an inhibitor of the sarcoplasmic reticulum Ca2+ pump, was used to decrease the rate of decline of [Ca2+]i after tetanic stimulation. The decay time courses of both [Ca2+]i and force were dose-dependently slowed by TBQ up to 5 μM; the instantaneous [Ca2+]i– force relations were nearly identical at ≥1 μM TBQ, suggesting that the change in [Ca2+]i was slow enough to reach equilibrium with force. The [Ca2+]i–force data obtained from the two types of experiments were consistent with the Hill curve using a Hill coefficient of 3.2–3.9 and [Ca2+]i for half activation (Ca50) of 1.5–1.7 μM. However, if fura dextran reacts with Ca2+ with a 2.5-fold greater Kd as previously estimated from the kinetic fitting (Konishi and Watanabe. 1995. J. Gen. Physiol. 106:1123–1150), Ca50 would be 3.7–4.2 μM. We also studied the [Ca2+]–force relation in skinned fibers under similar experimental conditions. The average Hill coefficient and Ca50 were estimated to be 3.3 and 1.8 μM, respectively. Although uncertainties remain about the precise levels of [Ca2+]i, we conclude that the steady state force is a 3rd to 4th power function of [Ca2+]i, and Ca50 is in the low micromolar range in intact frog muscle fibers, which is in reasonable agreement with results obtained from skinned fibers.

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Backward Movements of Cross-Bridges by Application of Stretch and by Binding of MgADP to Skeletal Muscle Fibers in the Rigor State as Studied by X-Ray Diffraction

Biophysical Journal ◽

10.1016/s0006-3495(99)77338-8 ◽

1999 ◽

Vol 76 (4) ◽

pp. 1770-1783 ◽

Cited By ~ 19

Author(s):

Yasunori Takezawa ◽

Duck-Sool Kim ◽

Masaki Ogino ◽

Yasunobu Sugimoto ◽

Takakazu Kobayashi ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Fibers ◽

X Ray Diffraction ◽

X Ray ◽

Rigor State ◽

Skeletal Muscle Fibers ◽

Cross Bridges

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Sarcomere-Length Dependence of the Low Angle X-Ray Pattern from Skeletal Muscle Fibers at Rest and during Isometric Contraction

Biophysical Journal ◽

10.1016/j.bpj.2011.11.806 ◽

2012 ◽

Vol 102 (3) ◽

pp. 147a-148a

Author(s):

Gabriella Piazzesi ◽

Massimo Reconditi ◽

Elisabetta Brunello ◽

Luca Fusi ◽

Marco Linari ◽

...

Keyword(s):

Skeletal Muscle ◽

Isometric Contraction ◽

Sarcomere Length ◽

Muscle Fibers ◽

X Ray ◽

Length Dependence ◽

Skeletal Muscle Fibers

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Comparative measurements of potassium and chloride with ion-sensitive microelectrodes and x-ray microanalysis in cultured skeletal muscle fibers

In Vitro ◽

10.1007/bf02620913 ◽

1985 ◽

Vol 21 (1) ◽

pp. 45-48 ◽

Cited By ~ 6

Author(s):

H. Acker ◽

F. Pietruschka ◽

K. Zierold

Keyword(s):

Skeletal Muscle ◽

Muscle Fibers ◽

X Ray ◽

Skeletal Muscle Fibers ◽

Ion Sensitive Microelectrodes

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Chloride currents from the transverse tubular system in adult mammalian skeletal muscle fibers

The Journal of General Physiology ◽

10.1085/jgp.201010496 ◽

2010 ◽

Vol 137 (1) ◽

pp. 21-41 ◽

Cited By ~ 30

Author(s):

Marino DiFranco ◽

Alvaro Herrera ◽

Julio L. Vergara

Keyword(s):

Skeletal Muscle ◽

Time Course ◽

Muscle Fibers ◽

Optical Data ◽

Mammalian Skeletal Muscle ◽

Chloride Currents ◽

Actual Distribution ◽

Transverse Tubular System ◽

Skeletal Muscle Fibers ◽

Tubular System

Chloride fluxes are the main contributors to the resting conductance of mammalian skeletal muscle fibers. ClC-1, the most abundant chloride channel isoform in this preparation, is believed to be responsible for this conductance. However, the actual distribution of ClC-1 channels between the surface and transverse tubular system (TTS) membranes has not been assessed in intact muscle fibers. To investigate this issue, we voltageclamped enzymatically dissociated short fibers using a two-microelectrode configuration and simultaneously recorded chloride currents (ICl) and di-8-ANEPPS fluorescence signals to assess membrane potential changes in the TTS. Experiments were conducted in conditions that blocked all but the chloride conductance. Fibers were equilibrated with 40 or 70 mM intracellular chloride to enhance the magnitude of inward ICl, and the specific ClC-1 blocker 9-ACA was used to eliminate these currents whenever necessary. Voltage-dependent di-8-ANEPPS signals and ICl acquired before (control) and after the addition of 9-ACA were comparatively assessed. Early after the onset of stimulus pulses, di-8-ANEPPS signals under control conditions were smaller than those recorded in the presence of 9-ACA. We defined as attenuation the normalized time-dependent difference between these signals. Attenuation was discovered to be ICl dependent since its magnitude varied in close correlation with the amplitude and time course of ICl. While the properties of ICl, and those of the attenuation seen in optical records, could be simultaneously predicted by model simulations when the chloride permeability (PCl) at the surface and TTS membranes were approximately equal, the model failed to explain the optical data if PCl was precluded from the TTS membranes. Since the ratio between the areas of TTS membranes and the sarcolemma is large in mammalian muscle fibers, our results demonstrate that a significant fraction of the experimentally recorded ICl arises from TTS contributions.

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