Extracellular matrix remodelling induced by alternating electrical and mechanical stimulations increases the contraction of engineered skeletal muscle tissues

Abstract

Engineered skeletal muscles are inferior to natural muscles in terms of contractile force, hampering their potential use in practical applications. One major limitation is that the extracellular matrix (ECM) not only impedes the contraction but also ineffectively transmits the forces generated by myotubes to the load. In the present study, ECM remodelling improves contractile force in a short time, and a coordinated, combined electrical and mechanical stimulation induces the desired ECM remodelling. Notably, the application of single and combined stimulations to the engineered muscles remodels the structure of their ECM networks, which determines the mechanical properties of the ECM. Myotubes in the tissues are connected in parallel and in series to the ECM. The stiffness of the parallel ECM must be low not to impede contraction, while the stiffness of the serial ECM must be high to transmit the forces to the load. Both the experimental results and the mechanistic model suggest that the combined stimulation through coordination reorients the ECM fibres in such a way that the parallel ECM stiffness is reduced, while the serial ECM stiffness is increased. In particular, 3 and 20 minutes of alternating electrical and mechanical stimulations increase the force by 18% and 31%, respectively.

Figure 1

Co-stimulation system and fascicle-inspired engineered skeletal muscle tissue (eSMT). (a,b) The fascicle-inspired eSMT formed a cylindrical shape with length of 6 mm and diameter of approximately 75 μm (a). The tissues were stained using immunofluorescence technique to visualize the striations of α-actinin, which is a marker of differentiation and contractility (b, reproduced from reference 40 with permission from the Mary Ann Liebert, Inc., New Rochelle, NY). Scale bars represent 5 mm in (a) and 10 μm in (b). (c) Schematic of the experimental setup used to apply coordinated electric and mechanical stimulation to the eSMT. The co-stimulation system consists of electrodes for applying the electric potential, a cantilever wire moved by a servomotor, and a laser micrometre to monitor the displacement of the cantilever. The eSMT is pulled sideways with the cantilever to stretch eSMT to the desired strain. Contractile force was quantified by measuring deformation of the cantilever whose bending stiffness is known. (d,e) Cross-sectional images of unstimulated eSMTs stained for collagen IV (red) and actin (green) in longitudinal (d) and transverse (e) directions. Scale bars represent 10 μm.

Figure 2

Coordination of the combined electric and mechanical stimulation and synergistic performance enhancement following 3 minutes of stimulation. (a) Four patterns of coordination: electrical (Elec) and mechanical (Mech) stimulations alone, and in-phase (In, 0° phase shift) and out-of-phase (Out, 180° phase shift) co-stimulations. (b) Improvement in the contractile force induced by the combined stimulation with different phase shifts. SEM, n = 10, 5, 10 and 5. (c) Improvement in the contractile force induced by stimulations with different frequencies. SEM, n = 3, 10, and 3. (d) Comparison of the performance improvements in the contractile force induced by the four patterns of stimulation in (a), the two single types of stimulation (Elec and Mech) and two co-stimulations (In and Out). SEM, n = 9, 11, 10 and 10. Out-of-phase co-stimulation increased the contractile force by 18% in 3 minutes. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3

Mechanistic model of eSMT force generation and transmission, and changes in the mechanical property induced by ECM remodelling. (a) Mechanistic model of eSMT consisting of an active element (AE), parallel passive element (Parallel PE), and serial passive element (Serial PE). (b) Images of collagen IV (red) and actin in the myotubes (green) of the eSMTs to measure the fibre orientation distribution of the ECM network (left). Images of well-aligned collagen fibres (middle) and less-aligned fibres (right). Scale bars represent 5 μm. (c) Schematics showing the regions of the parallel and serial ECMs relative to the myotubes, and ECM fibre orientation θ measured from the longitudinal direction of myotube. (d,e) Orientation distribution of collagen IV in the parallel (d) and serial (e) ECM regions. Most ECM fibres were aligned parallel to the longitudinal direction of the myotubes (0°) by pretension. SEM, n = 8, 12, 11, and 10 (parallel), n = 3, 7, 3, and 7 (serial). (f) Comparison of fibre orientation factors (η_o) to predict the elastic modulus of the ECM network (E_L). The out-of-phase co-stimulation induced the highest elastic modulus (largest η_o) for the Serial PE and a low elastic modulus for the Parallel PE. SEM, n is the same as (d,e). *P < 0.05, **P < 0.01, and ns, not significant. (g) Schematic depicting the mechanism underlying the changes in performance induced by ECM remodelling following the application of the out-of-phase co-stimulation. The out-of-phase co-stimulation decreases the stiffness of the parallel ECM for less impedance on muscle contraction and increases the stiffness of the serial ECM to increase force transmission to the load.

Figure 4

Investigation of changes in the active element and long-term (20 minutes) effects of the stimulation. (a) Measurement of sarcomere length based on the intensity of α-actinin immunostaining images. The schematic shows the sarcomere structure and length. (b) Percentage (%) distributions of sarcomere length in each range after applying the electric potential (Elec), mechanical stretching (Mech), in-phase co-stimulation (In), and out-of-phase co-stimulation (Out) for 3 minutes. (c) Average sarcomere length after applying the four different types of stimulations: Elec, Mech, In, and Out. SD, n = 69, 96, 64, and 145. (d) Longer-term performance enhancement of eSMTs following the application of the four stimulations for 20 minutes, SEM, n = 3 for all. Out-of-phase co-stimulation enhanced the contractile force by 31% in 20 minutes.