Acute exposure to extracellular BTP2 does not inhibit Ca2+ release during EC coupling in intact skeletal muscle fibers

The inhibitor of store-operated Ca2+ entry (SOCE) BTP2 was reported to inhibit ryanodine receptor Ca2+ leak and electrically evoked Ca2+ release from the sarcoplasmic reticulum when introduced into mechanically skinned muscle fibers. However, it is unclear how effects of intracellular application of a highly lipophilic drug like BTP2 on Ca2+ release during excitation–contraction (EC) coupling compare with extracellular exposure in intact muscle fibers. Here, we address this question by quantifying the effect of short- and long-term exposure to 10 and 20 µM BTP2 on the magnitude and kinetics of electrically evoked Ca2+ release in intact mouse flexor digitorum brevis muscle fibers. Our results demonstrate that neither the magnitude nor the kinetics of electrically evoked Ca2+ release evoked during repetitive electrical stimulation were altered by brief exposure (2 min) to either BTP2 concentration. However, BTP2 did reduce the magnitude of electrically evoked Ca2+ release in intact fibers when applied extracellularly for a prolonged period of time (30 min at 10 µM or 10 min at 20 µM), consistent with slow diffusion of the lipophilic drug across the plasma membrane. Together, these results indicate that the time course and impact of BTP2 on Ca2+ release during EC coupling in skeletal muscle depends strongly on whether the drug is applied intracellularly or extracellularly. Further, these results demonstrate that electrically evoked Ca2+ release in intact muscle fibers is unaltered by extracellular application of 10 µM BTP2 for <25 min, validating this use to assess the role of SOCE in the absence of an effect on EC coupling.

Download Full-text

Measurement of force and calcium release using mechanically skinned fibers from mammalian skeletal muscle

Journal of Applied Physiology ◽

10.1152/japplphysiol.00445.2018 ◽

2018 ◽

Vol 125 (4) ◽

pp. 1105-1127 ◽

Cited By ~ 15

Author(s):

Graham D. Lamb ◽

D. George Stephenson

Keyword(s):

Skeletal Muscle ◽

Muscle Fiber ◽

Calcium Release ◽

Muscle Fibers ◽

Mammalian Skeletal Muscle ◽

Ec Coupling ◽

Skeletal Muscle Fibers ◽

Intact Muscle ◽

Key Variables ◽

Coupling Sequence

The mechanically skinned (or “peeled”) skeletal muscle fiber technique is a highly versatile procedure that allows controlled examination of each of the steps in the excitation-contraction (EC)-coupling sequence in skeletal muscle fibers, starting with excitation/depolarization of the transverse tubular (T)-system through to Ca2+ release from sarcoplasmic reticulum (SR) and finally force development by the contractile apparatus. It can also show the overall response of the whole EC-coupling sequence together, such as in twitch and tetanic force responses. A major advantage over intact muscle fiber preparations is that it is possible to set and rapidly manipulate the “intracellular” conditions, allowing examination of the effects of key variables (e.g., intracellular pH, ATP levels, redox state, etc.) on each individual step in EC coupling. This Cores of Reproducibility in Physiology (CORP) article describes the rationale, procedures, and experimental details of the various ways in which the mechanically skinned fiber technique is used in our laboratory to examine the physiological mechanisms controlling Ca2+ release and contraction in skeletal muscle fibers and the aberrations and dysfunction occurring with exercise and disease.

Download Full-text

Recordings of action potentials, charge movements, and sarcoplasmic reticulum Ca2+ release in isolated adult zebrafish fast skeletal muscle fibers reveals very fast kinetics of excitation–contraction coupling

The Journal of General Physiology ◽

10.1085/jgp.2021ecc14 ◽

2021 ◽

Vol 154 (9) ◽

Author(s):

Romane Idoux ◽

Christine Berthier ◽

Vincent Jacquemond ◽

Bruno Allard

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Action Potentials ◽

Muscle Fibers ◽

Fast Skeletal Muscle ◽

Fast Kinetics ◽

Ec Coupling ◽

Skeletal Muscle Fibers ◽

Charge Movements ◽

Kinetics Of

The zebrafish has emerged as a very relevant animal model to decipher the pathophysiology of human muscle disorders. However, the vast majority of studies on zebrafish skeletal muscle have investigated genetic, histological, and molecular aspects, but functional approaches at the cellular level, especially in the field of excitation–contraction (EC) coupling, are scarcer and generally limited to cultured myotubes or fibers from embryonic zebrafish. Considering that zebrafish undergoes profound metamorphosis during transition from larval to adult stage and that number of muscle pathologies come up at ages far beyond embryonic stages, there is an actual need to investigate EC coupling in fully differentiated zebrafish skeletal muscle. In the present study, we were able to implement current and voltage clamp combined with intracellular Ca2+ measurements using the intracellularly loaded Ca2+ dye indo-1 in enzymatically isolated fast skeletal muscle fibers from 1-yr old zebrafish. Recording of action potentials (AP) in current-clamp conditions revealed very fast kinetics of the repolarization phase of AP. Measurements of intramembrane charge movements in voltage-clamp conditions showed that charge movement density was half that measured in mammalian fibers, but they displayed much faster kinetics. Ca2+ transients elicited by depolarization displayed a voltage-dependent phase of activation and voltage- and time-dependent phase of inactivation. Recording of Ca2+ signals elicited by trains of AP at different rates in current-clamp conditions indicated that Ca2+ signals fused at very high stimulation frequencies with no sign of Ca2+ signal decay for the entire 0.5 s duration of the stimulation, giving evidence that fibers were still able to generate AP and the sarcoplasmic reticulum to release Ca2+ with stimulation rates as high as 200 Hz. These data indicate that adult zebrafish fast skeletal muscle fibers exhibit strikingly fast kinetics of EC coupling from AP firing to charge movements and sarcoplasmic reticulum Ca2+ release.

Download Full-text

Myosin regulatory light chain modulates the Ca2+ dependence of the kinetics of tension development in skeletal muscle fibers

Biophysical Journal ◽

10.1016/s0006-3495(96)79799-0 ◽

1996 ◽

Vol 70 (5) ◽

pp. 2333-2340 ◽

Cited By ~ 36

Author(s):

J.R. Patel ◽

G.M. Diffee ◽

R.L. Moss

Keyword(s):

Skeletal Muscle ◽

Light Chain ◽

Muscle Fibers ◽

Regulatory Light Chain ◽

Myosin Regulatory Light Chain ◽

Tension Development ◽

Skeletal Muscle Fibers ◽

Kinetics Of

Download Full-text

Disassembly kinetics of thick filaments in rabbit skeletal muscle fibers. Effects of ionic strength, Ca2+ concentration, pH, temperature, and cross-bridges on the stability of thick filament structure

Biophysical Journal ◽

10.1016/s0006-3495(85)83916-3 ◽

1985 ◽

Vol 47 (3) ◽

pp. 267-275 ◽

Cited By ~ 7

Author(s):

H. Higuchi ◽

S. Ishiwata

Keyword(s):

Skeletal Muscle ◽

Ionic Strength ◽

Muscle Fibers ◽

Thick Filament ◽

Rabbit Skeletal Muscle ◽

Thick Filaments ◽

Filament Structure ◽

Cross Bridges ◽

The Stability ◽

Kinetics Of

Download Full-text

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.

Download Full-text

Determinants of relaxation rate in skinned frog skeletal muscle fibers

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.6.c1608 ◽

1998 ◽

Vol 274 (6) ◽

pp. C1608-C1615 ◽

Cited By ~ 17

Author(s):

Philip A. Wahr ◽

J. David Johnson ◽

Jack. A. Rall

Keyword(s):

Skeletal Muscle ◽

Muscle Fibers ◽

Slow Phase ◽

Frog Skeletal Muscle ◽

Force Of Contraction ◽

Thin Filaments ◽

Fast Phase ◽

Skeletal Muscle Fibers ◽

Kinetics Of ◽

Overall Kinetics

The influences of sarcomere uniformity and Ca2+ concentration on the kinetics of relaxation were examined in skinned frog skeletal muscle fibers induced to relax by rapid sequestration of Ca2+ by the photolysis of the Ca2+ chelator, diazo-2, at 10°C. Compared with an intact fiber, diazo-2-induced relaxation exhibited a faster and shorter initial slow phase and a fast phase with a longer tail. Stabilization of the sarcomeres by repeated releases and restretches during force development increased the duration of the slow phase and slowed its kinetics. When force of contraction was decreased by lowering the Ca2+concentration, the overall kinetics of relaxation was accelerated, with the slow phase being the most sensitive to Ca2+ concentration. Twitchlike contractions were induced by photorelease of Ca2+ from a caged Ca2+ (DM-Nitrophen), with subsequent Ca2+ sequestration by intact sarcoplasmic reticulum or Ca2+ rebinding to caged Ca2+. These twitchlike responses exhibited relaxation kinetics that were about twofold slower than those observed in intact fibers. Results suggest that the slow phase of relaxation is influenced by the degree of sarcomere homogeneity and rate of Ca2+ dissociation from thin filaments. The fast phase of relaxation is in part determined by the level of Ca2+ activation.

Download Full-text

Parvalbumin relaxes frog skeletal muscle when sarcoplasmic reticulum Ca(2+)-ATPase is inhibited

AJP Cell Physiology ◽

10.1152/ajpcell.1996.270.2.c411 ◽

1996 ◽

Vol 270 (2) ◽

pp. C411-C417 ◽

Cited By ~ 170

Author(s):

Y. Jiang ◽

J. D. Johnson ◽

J. A. Rall

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Relaxation Rate ◽

Time Course ◽

Muscle Fibers ◽

Peak Force ◽

Half Time ◽

Frog Skeletal Muscle ◽

Twitch Force ◽

Total Failure

Inhibition of sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase) with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBQ) in frog skeletal muscle fibers at 10 degrees C prolonged the half time of the fall of the Ca2+ transient by 62% and twitch force by 100% and increased peak force by 120% without increasing the amplitude of the Ca2+ signal. In the presence of TBQ the rate of relaxation and the rate of fall of Ca2+ became progressively slower in a series of twitches until relaxation failed. Relaxation rate decreased with a time course (approximately 2 s-1) similar to the Mg2+ off rate from purified parvalbumin (PA; 3.6 s-1). TBQ slowed the rate of fall of Ca2+ (5-fold) and force (8-fold) in a 0.3-s tetanus so that the rate of fall of Ca2+ (approximately 2.5 s-1) was similar to the Mg2+ off rate from PA. TBQ caused a near total failure of both Ca2+ sequestration and relaxation in a 1.1-s tetanus, during which PA would be saturated with Ca2+ and could not contribute to relaxation. Thus, when the SR Ca(2+)-ATPase is inhibited, Mg(2+)-PA can sequester Ca2+ and produce relaxation at a rate that is defined by the Mg2+ off rate from PA.

Download Full-text