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

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

Mechanisms of Calcium Release in Skinned Mammalian Skeletal Muscle Fibers

Calcium in Biological Systems ◽

10.1007/978-1-4613-2377-8_37 ◽

1985 ◽

pp. 331-337

Author(s):

Sue K. Bolitho Donaldson ◽

Robert Dunn ◽

Daniel A. Huetteman

Keyword(s):

Skeletal Muscle ◽

Calcium Release ◽

Muscle Fibers ◽

Mammalian Skeletal Muscle ◽

Skeletal Muscle Fibers

Download Full-text

Involvement of calpains in Ca2+-induced disruption of excitation-contraction coupling in mammalian skeletal muscle fibers

AJP Cell Physiology ◽

10.1152/ajpcell.00008.2009 ◽

2009 ◽

Vol 296 (5) ◽

pp. C1115-C1122 ◽

Cited By ~ 26

Author(s):

Esther Verburg ◽

Robyn M. Murphy ◽

Isabelle Richard ◽

Graham D. Lamb

Keyword(s):

Skeletal Muscle ◽

Surface Membrane ◽

Muscle Fibers ◽

Mammalian Skeletal Muscle ◽

Skinned Muscle ◽

Ec Coupling ◽

Calpain 3 ◽

Skeletal Muscle Fibers ◽

Longus Muscle ◽

Rate Of Decline

In skeletal muscle fibers, the coupling between excitation of the surface membrane and the release of Ca2+ from the sarcoplasmic reticulum is irreversibly disrupted if cytoplasmic Ca2+ concentration ([Ca2+]) is raised to micromolar levels for a prolonged period. This excitation-contraction (EC) uncoupling may contribute to muscle weakness after some types of exercise and in certain muscle diseases and has been linked to structural alteration of the triad junctions, but its molecular basis is unclear. Both μ-calpain, a ubiquitous Ca2+-activated protease, and muscle-specific calpain-3 become autolytically activated at micromolar Ca2+ and have been suggested to be responsible for the uncoupling. This study used controlled Ca2+ exposure in mechanically skinned fibers from extensor digitorum longus muscle to show that EC uncoupling still occurs in muscle fibers of calpain-3-deficient mice, with a Ca2+ dependence indistinguishable from that in normal mice and rats. Western blotting of muscle fibers that had been partially EC uncoupled by exposure to an intermediate Ca2+ level (∼5 μM Ca2+ for 3 min, no ATP) showed the presence of autolytic activation of a proportion of the μ-calpain present, but with little or no activation of calpain-3. Homogenates of normal and calpain-3-deficient muscles exposed to micromolar Ca2+ displayed similar levels of diffusible proteolytic activity, as gauged by the rate of decline of passive force in stretched, skinned muscle fibers. Exogenously added μ-calpain, preactivated by elevated [Ca2+] and applied in the presence of 1 μM Ca2+, disrupted EC coupling in a manner similar to raised [Ca2+]. We conclude that calpain-3 is not responsible for Ca2+-induced disruption of EC coupling, but that μ-calpain is a plausible candidate.

Download Full-text

Desflurane Induces Only Minor Ca2+Release from the Sarcoplasmic Reticulum of Mammalian Skeletal Muscle

Anesthesiology ◽

10.1097/00000542-200009000-00034 ◽

2000 ◽

Vol 93 (3) ◽

pp. 832-836 ◽

Cited By ~ 9

Author(s):

Gudrun Kunst ◽

Astrid G. Stucke ◽

Bernhard M. Graf ◽

Eike Martin ◽

Rainer H. A. Fink

Keyword(s):

Skeletal Muscle ◽

Calcium Release ◽

Muscle Fibers ◽

Mammalian Skeletal Muscle ◽

Skinned Muscle ◽

Skeletal Muscle Fibers ◽

Force Relation ◽

The Individual ◽

Few Data ◽

Sensitizing Effect

Background Desflurane is a weaker trigger of malignant hyperthermia than is halothane. There are very few data of the pathophysiologic background of this observation. Therefore, the authors' aim was to investigate the direct effect of desflurane on calcium release in skinned skeletal muscle fibers. Methods For the measurements, single saponin-skinned muscle fiber preparations of BALB/c mice were used. For Ca2+ release experiments, liquid desflurane at 0.6 and 3.5 mm was applied to weakly calcium-buffered solutions with no added Ca2+. Desflurane was diluted in strongly Ca2+-buffered solutions, with [Ca2+] between 3.0 and 24.9 micrometer for [Ca2+]-force relations. Force transients were transformed into Ca2+ transients based on the individual [Ca2+]-force relations. As controls, 30 mm caffeine and equimolar sevoflurane were investigated in the same muscle fibers. Results At 3.5 mm, desflurane induced peak force transients of 8 +/- 4% (mean +/- SD) of maximal Ca2+-activated force (Tmax). These peak values were significantly smaller than those in the presence of 3.5 mm sevoflurane (24 +/- 10% of Tmax, P < 0.05), and 4 or 5 times smaller than previously reported Ca2+-release-induced force transients by equimolar halothane. Calculated peak Ca2+ transients derived from force transients and induced by 3.5 and 0.6 mm desflurane were significantly smaller than those induced by 30 mm caffeine. The [Ca2+]-force relation was shifted by desflurane, resulting in a Ca2+-sensitizing effect. The maximal Ca2+-activated force was significantly increased by 0.6 mm desflurane in comparison with the control, with no added substance (P </= 0.05). Conclusion Desflurane induces only slight Ca2+ release in skinned skeletal muscle fibers.

Download Full-text

The Adaptive Potential of Skeletal Muscle Fibers

Canadian Journal of Applied Physiology ◽

10.1139/h02-023 ◽

2002 ◽

Vol 27 (4) ◽

pp. 423-448 ◽

Cited By ~ 109

Author(s):

Dirk Pette

Keyword(s):

Skeletal Muscle ◽

Fiber Type ◽

Muscle Fibers ◽

Protein Isoforms ◽

Mammalian Skeletal Muscle ◽

Adaptive Potential ◽

Hybrid Fibers ◽

Neuromuscular Activity ◽

Skeletal Muscle Fibers ◽

Initial Location

Mammalian skeletal muscle fibers display a great adaptive potential. This potential results from the ability of muscle fibers to adjust their molecular, functional, and metabolic properties in response to altered functional demands, such as changes in neuromuscular activity or mechanical loading. Adaptive changes in the expression of myofibrillar and other protein isoforms result in fiber type transitions. These transitions occur in a sequential order and encompass a spectrum of pure and hybrid fibers. Depending on the quality, intensity, and duration of the alterations in functional demand, muscle fibers may undergo functional transitions in the direction of slow or fast, as well as metabolic transitions in the direction of aerobic-oxidative or glycotytic. The maximum range of possible transitions in either direction depends on the fiber phenotype and is determined by its initial location in the fiber spectrum. Key words: Ca-sequestering proteins, energy metabolism, fiber type transition, myofibrillar protein isofonns, myosin, neuromuscular activity

Download Full-text