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. 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 µN ± 0.15 µN and tetanic forces (100 Hz stimulation frequency) of 2.38 µN ± 0.71 µN, corresponding to a tension of 0.44 kPa ± 0.25 kPa and 2.53 kPa ± 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 and high throughput 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. Statement of significanceWe describe a high speed, high throughput method for the simultaneous measurement of contractile force and cytoplasmic calcium dynamics following electrical pulse stimulation of muscle fibers embedded in a 3-dimensional biopolymer matrix. In contrast to the classical approach of attaching muscle fibers to a force-transducer, our method allows for a highly efficient, parallel analysis of large numbers of fibers under different treatment conditions.

Nonlinear elasticity of biological basement membrane revealed by rapid inflation and deflation

“…Furthermore, our in situ measurement reveals marked nonlinear elasticity of the BMs whose onset is below 10% of strain. This behavior is in direct contrast to the large linear elastic regime observed in reconstituted Matrigel (24,25) yet is consistent with a wide variety of biological tissues and biopolymer networks (26,40,41). This nonlinear stiffening behavior of the BM may have an important role in maintaining tissue mechanical integrity during growth and deformation (e.g., to avoid structural instability), which is a classical behavior leading to drastic expansion or even rupture when inflating most elastomeric balloons.…”

“…With a slight increase of strain, the tangent modulus K of BM increases by several times for both uniaxial and biaxial deformation. This is in direct contrast to the wide linear elastic regime observed on reconstituted Matrigel (24,25), indicating distinct structural characteristics between natural BMs and Matrigel reconstituted from BM extracts. Indeed, clear fibrous network structures can be observed in the intact BMs, as shown by the TEM images in Fig.…”

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

“…Furthermore, micro-tissues show a reduced force-frequency relationship compared to 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 to a factor of 6.7 in adult SOL fibers, a factor of 3.8 in EDL fibers (37), and a factor of 5.7 in FDB fibers (42).…”

“…Contractile isometric twitch tension of single adult murine SOL and EDL fibers have been reported to be approximately 50 kPa and 100 kPa, respectively (41). By contrast, when primary isolated mouse flexor digitorum brevis (FDB) fibers are embedded in a soft 200 Pa (Young’s modulus) Matrigel, they generate low tension comparable to that of our micro-tissues (twitch tension 0.44 kPa at 1 Hz, tetanic tension 2.53 kPa at 100 Hz) (42). It is currently unknown whether this low tension in these primary FDB fibers is the result of excessive fiber shortening of more than 40% or due to other reasons such as cell adhesion to Matrigel.…”

Global and local tension measurements in biomimetic skeletal muscle tissues reveals early mechanical homeostasis

“…Recently, a method to determine contraction forces of myotubes grown within collagen or other ECM-like matrices using 3D traction force microscopy was described Rausch et al (2019). This approach resolves force generation not only at the endpoints of the myotubes, as in the post systems, but can also determine traction forces along the length of the myotube.…”