Dataset Information

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 ± 0.15 ?N and tetanic forces (100-Hz stimulation frequency) of 2.38 ± 0.71 ?N, corresponding to a tension of 0.44 ± 0.25 kPa and 2.53 ± 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.

SUBMITTER: Rausch M

PROVIDER: S-EPMC7002922 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Rausch Martin M Böhringer David D Steinmann Martin M Schubert Dirk W DW Schrüfer Stefan S Mark Christoph C Fabry Ben B

Biophysical journal 20191224 3

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 de ...[more]

PMID: 31952805

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Project description:Skeletal muscle can adapt to increased mechanical loads by undergoing hypertrophy. Transient reductions in whole muscle force production have been reported during the onset of hypertrophy, but contractile changes in individual muscle fibers have not been previously studied. Additionally, the extracellular matrix (ECM) stores and transmits forces from muscle fibers to tendons and bones, and determining how the ECM changes during hypertrophy is important in understanding the adaptation of muscle tissue to mechanical loading. Using the synergist ablation model, we sought to measure changes in muscle fiber contractility, collagen content, and cross-linking, and in the expression of several genes and activation of signaling proteins that regulate critical components of myogenesis and ECM synthesis and remodeling during muscle hypertrophy. Tissues were harvested 3, 7, and 28 days after induction of hypertrophy, and nonoverloaded rats served as controls. Muscle fiber specific force (sFo), which is the maximum isometric force normalized to cross-sectional area, was reduced 3 and 7 days after the onset of mechanical overload, but returned to control levels by 28 days. Collagen abundance displayed a similar pattern of change. Nearly a quarter of the transcriptome changed over the course of overload, as well as the activation of signaling pathways related to hypertrophy and atrophy. Overall, this study provides insight into fundamental mechanisms of muscle and ECM growth, and indicates that although muscle fibers appear to have completed remodeling and regeneration 1 mo after synergist ablation, the ECM continues to be actively remodeling at this time point.NEW & NOTEWORTHY This study utilized a rat synergist ablation model to integrate changes in single muscle fiber contractility, extracellular matrix composition, activation of important signaling pathways in muscle adaption, and corresponding changes in the muscle transcriptome to provide novel insight into the basic biological mechanisms of muscle fiber hypertrophy.

Project description:Tractions exerted by cells on their surroundings play an important role in many biological processes including stem cell differentiation, tumorigenesis, cell migration, cancer metastasis, and angiogenesis. The ability to quantify these tractions is important in understanding and manipulating these processes. Three-dimensional traction force microscopy (3DTFM) provides reliable means of evaluating cellular tractions by first measuring the displacement of fluorescent beads in response to these tractions in the surrounding matrix, and then using this measurement to compute the tractions. However, most applications of 3DTFM assume that the surrounding extra-cellular matrix (ECM) is non-fibrous, despite the fact that in many natural and synthetic environments the ECM contains a significant proportion of fibrous components. Motivated by this, we develop a computational approach for determining tractions, while accounting for the fibrous nature of the ECM. In particular, we make use of a fiber-based constitutive model in which the stress contains contributions from a distribution of nonlinear elastic fibers and a hyperelastic matrix. We solve an inverse problem with the nodal values of the traction vector as unknowns, and minimize the difference between a predicted displacement field, obtained by solving the equations of equilibrium in conjunction with the fiber-based constitutive model, and the measured displacement field at the bead locations. We employ a gradient-based minimization method to solve this problem and determine the gradient efficiently by solving for the appropriate adjoint field. We apply this algorithm to problems with experimentally observed cell geometries and synthetic, albeit realistic, traction fields to gauge its sensitivity to noise, and quantify the impact of using an incorrect constitutive model: the so-called model error. We conclude that the approach is robust to noise, yielding about 10% error in tractions for 5% displacement noise. We also conclude that the impact of model error is significant, where using a nonlinear exponential hyperelastic model instead of the fiber-based model, can lead to more than 100% error in the traction field. These results underline the importance of using appropriate constitutive models in 3DTFM, especially in fibrous ECM constructs.

Dataset Information

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

Publications

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

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