Measurement of Skeletal Muscle Fiber Contractility with High-Speed Traction Microscopy

Abstract

We describe a technique for simultaneous quantification of the contractile forces and cytosolic calcium dynamics of muscle fibers embedded in three-dimensional biopolymer gels under auxotonic loading conditions. We derive a scaling law for linear elastic matrices such as basement membrane extract hydrogels (Matrigel) that allows us to measure contractile force from the shape of the relaxed and contracted muscle cell and the Young's modulus of the matrix without further knowledge of the matrix deformations surrounding the cell and without performing computationally intensive inverse force reconstruction algorithms. We apply our method to isolated mouse flexor digitorum brevis (FDB) fibers that are embedded in 10 mg/mL Matrigel. Upon electrical stimulation, individual FDB fibers show twitch forces of 0.37 5 0.15 mN and tetanic forces (100-Hz stimulation frequency) of 2.38 5 0.71 mN, corresponding to a tension of 0.44 5 0.25 kPa and 2.53 5 1.17 kPa, respectively. Contractile forces of FDB fibers increase in response to caffeine and the troponin-calcium stabilizer tirasemtiv, similar to responses measured in whole muscle. From simultaneous high-speed measurements of cell length changes and cytosolic calcium concentration using confocal line scanning at a frequency of 2048 Hz, we show that twitch and tetanic force responses to electric pulses follow the low-pass filtered calcium signal. In summary, we present a technically simple high-speed method for measuring contractile forces and cytosolic calcium dynamics of single muscle fibers. We expect that our method will help to reduce preparation time, costs, and the number of sacrificed animals needed for experiments such as drug testing.

The desmin mutation R349P increases contractility and fragility of stem cell‐generated muscle micro‐tissues

“…Micro‐tissues show a reduced force‐frequency relationship compared with primary skeletal muscle, for currently unknown reasons. At a pacing frequency of 100 Hz, the contractile force of the micro‐tissues increased only by a factor of around 1.7, compared with a factor of 6.7 in adult SOL fibres, a factor of 3.8 in EDL fibres [38], and a factor of 5.7 in FDB fibres [41]. Furthermore, micro‐tissues generate static forces that are comparable in magnitude to the active force, whereas static forces (also called residual tension, or tonus) in isolated primary skeletal muscle fibres are largely absent.…”

Calcium measurements in enzymatically dissociated or mechanically microdissected mouse primary skeletal muscle fibers

“…For measurements of calcium in enzymatically dissociated fibers, we refer to the interested reader to the accurate methodological descriptions in ref. 10 , 11 Note that the following parts IIIa and IIIb are alternatives for calcium measurements. In our experience, the best results are obtained with injection of the dye.…”

Contractile force assessment methods for in vitro skeletal muscle tissues

“…However, it presents some limitations inherent to the setup, such as the requirement of relatively low cell densities to track forces correctly, which may impact the size of de novo generated myotubes ( Bruegmann et al, 2010 ). Thus, some authors use TFM with isolated native muscle fibers to overcome this limitation ( Rausch et al, 2020 ). Inspired by this method other authors have determined the tensional status of hydrogels-containing human or C2C12 myotubes, by tracking deformable polyacrylamide fluorescent microbeads throughout the culture compaction and development ( Hofemeier et al, 2021 ), or by detecting deformation of the substrate through Particle Imaging Velocity ( Zhang et al, 2018 ; Cheesbrough et al, 2022 ).…”