Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Summary

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca²⁺ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

Abstract

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca²⁺ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca²⁺ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Introduction

Skeletal muscles attach to bones of the skeleton and generate contractile forces under the control of the central nervous system. Excitation-Contraction coupling (ECC) refers to the process of converting an electrical stimulus to a mechanical response. Ca²⁺ signaling is an essential component of the contractile function in skeletal muscle. Effective Ca²⁺ mobilization from sarcoplasmic reticulum (SR) is an important component for ECC in muscle cells^{1, 2}, and changes in intracellular Ca²⁺ signaling underlie the corresponding contractile dysfunction in a number of muscle diseases^3-5. Pro....

Protocol

Solution composition:

2.5 mM Ca²⁺ Tyrode solution: 140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2.5 mM CaCl₂, 2 mM MgCl₂ and 10 mM glucose

0 mM Ca²⁺ Tyrode solution: 140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM MgCl₂, 0.1 mM ethylene glycol tetraacetic acid (EGTA) and 10 mM glucose

Note: bathing solution should be saturated with 100% O₂ if using the above solution, but with 95% O₂ with 5% CO₂ if using bicarbonate-based buffers to keep pH constant. 2.5 mM Ca²⁺ is added to the bathing buffer to recapitulat....

Discussion

Measurement of contractile force and fatigability is important for the overall evaluation of skeletal muscle function. The major purpose of this assay is to identify changes in muscle force and fatiguing properties under certain pathological conditions, such as sarcopenia and muscle fatigue, and to test the effect of drugs/reagents on muscle contractility. Since the muscle force is closely related with intracellular Ca²⁺ release, extracellular Ca²⁺ entry and the crosstalk between these two, we can a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-APB	Tocris	1224	Blocker of a number of Ca2+ entry channels including SOC and TRP etc.
SKF96365	Sigma	SKF-96365	Blocker of a number of Ca2+ entry channels including SOC and receptor-mediated Ca2+ entry etc.
BTP-2	Millipore	203890-5MG	Relatively specific SOC blocker
CPA	Sigma	C1530	Reversible SERCA blocker
caffeine	Sigma	C0750	Fast action RyR agonist
Radnoti Four Unit Tissue Organ Bath System	Radnoti	159920
Combination Tissue Support/Stimulating Electrode	Radnoti	160151	Vertical Zig Zag Type with tissue support
Quad Bridge Amp	ADInstruments	FE224
PowerLab/400	ADInstruments		This product is no longer available. Choose other version of the data acquisition system.
Force Transducers (5 mg - 25 g)	ADInstruments	MLT0201/RAD
Chart v4.02	ADInstruments		LabChart 7.3 is the latest version of Chart software.
S8800 Dual Pulse Digital Stimulator	GRASS TECHNOLOGIES		This product is no longer available. S88X Dual Output Square Pulse Stimulator is a newer stimulator.
RF Transformer Isolation Unit	GRASS TECHNOLOGIES	Model SIU5