Myosin cross-bridge kinetics slow at longer muscle lengths during isometric contractions in intact soleus from mice

Abstract

Muscle contraction results from force-generating cross-bridge interactions between myosin and actin. Cross-bridge cycling kinetics underlie fundamental contractile properties, such as active force production and energy utilization. Factors that influence cross-bridge kinetics at the molecular level propagate through the sarcomeres, cells and tissue to modulate whole-muscle function. Conversely, movement and changes in the muscle length can influence cross-bridge kinetics on the molecular level. Reduced, single-molecule and single-fibre experiments have shown that increasing the strain on cross-bridges may slow their cycling rate and prolong their attachment duration. However, whether these strain-dependent cycling mechanisms persist in the intact muscle tissue, which encompasses more complex organization and passive elements, remains unclear. To investigate this multi-scale relationship, we adapted traditional step-stretch protocols for use with mouse soleus muscle during isometric tetanic contractions, enabling novel estimates of length-dependent cross-bridge kinetics in the intact skeletal muscle. Compared to rates at the optimal muscle length (L_o), we found that cross-bridge detachment rates increased by approximately 20% at 90% of L_o (shorter) and decreased by approximately 20% at 110% of L_o (longer). These data indicate that cross-bridge kinetics vary with whole-muscle length during intact, isometric contraction, which could intrinsically modulate force generation and energetics, and suggests a multi-scale feedback pathway between whole-muscle function and cross-bridge activity.

Keywords: myosin cross-bridges intact muscle.

Figure 1.

Tetanic stress of electrically stimulated mouse soleus muscle. (a) Average muscle stress (solid lines) is plotted against time for three muscle lengths: optimal length (L_o) and ±10% MTU length of L_o (shaded regions represent SEM for each length). Tetanic contractions were elicited for 1 s via 20 mV stimuli at 100 Hz, with example data shown solely for 17°C. (b) Maximal stress of tetanically stimulated muscle at three muscle lengths for 17°C and 27°C. (c) Maximal stress was determined as average stress over the final 20 ms before the end of electrical excitation. Asterisks denote differences from the measured value at L_o within a temperature (p < 0.05), and the dagger denotes differences between temperatures for the same muscle length. n = 10 muscles.

Figure 2.

Composite force–length relationship from 10 MTUs. Each MTU was stimulated at 20 V and 100 Hz of 0.2 ms pulse width for 1 s. Each colour represents a single MTU from a single mouse. Force measurements were made at 5–7 lengths from each muscle. Dashed line represents a fourth-order polynomial fit to the composite data [22].

Figure 3.

Protocol for step-stretch and modulus response of an example muscle during tetanic muscle contraction. Upper trace: motor position is plotted against time, where the MTU was stretched 1% of the total MTU length and then held at that longer length to enable measurement of the muscle stress–strain relationship. Middle trace: active muscle stress (σ) was plotted against time; muscle stress increased toward maximal isometric stress as the muscle was stimulated, and then stress briefly spiked with the step-stretch change (i.e. the strain stimulus initiated at time t = t₀), followed by stress decaying towards a new steady-state value at the longer MTU length. The open circle at σ₀ denotes muscle stress at time t = t₀, representing the tetanic stress value when the step-stretch was applied. Maximal stress is denoted by σ₁ (open circle), and the nadir of the subsequent stress response is denoted by σ₂ (open circle). The closed circle at σ_t1/2 represents the time-point and stress value 50% of the way between σ₁ and σ₂. Left inset: enhanced time scale of the rise and fall phase surrounding the step-stretch. Right inset: the modulus response as function of time (Y(t) = stress (t)/strain (t)), decaying from the maximum value at σ₁, was fitted to equation (2.2) to estimate kinetic parameters. Bottom trace: passive muscle stress plotted against the same time scale with a step-stretch protocol, but in the absence of electrical stimulation.

Figure 4.

Temperature dependence of the modulus following a step-stretch. Solid black lines show the average modulus response plotted against time response at 17°C and 27°C. Temperature-dependent differences in cross-bridge cycling underlie the different dynamics shown between these two traces. Dashed lines represent fits of these average data to equation (2.2), with average parameter values from the fits listed in table 1. n = 10 muscles.

Figure 5.

Effects of MTU length on modulus following a step-stretch at (a) 17°C and (b) 27°C. Solid lines show the average modulus response plotted against time for a given length, and dashed lines represent fits of these average data to equation (2.2). Length-dependent differences in cross-bridge cycling underlie the different dynamics shown by the blue, black and red traces within each panel. n = 10 muscles. (Online version in colour.)

Figure 6.

Moduli values were fitted to equation (2.2) to provide estimates of tissue viscoelasticity, cross-bridge binding and cross-bridge kinetics. (a,b) Parameter A represents the combined mechanical stress of the muscle, while parameter k describes the viscoelasticity of these passive elements (0 = purely elastic response, 1 = purely viscous response). (c,d) Parameter B represents the magnitude of work-producing muscle mechanics, with parameter 2πb representing the cross-bridge recruitment rate. (e,f) Parameter C represents the magnitude of work-absorbing muscle mechanics, with parameter 2πc representing the cross-bridge detachment rate. Asterisks denote differences from the measured value at L_o within a temperature (p < 0.05), and daggers denote differences between temperatures for the same muscle length. n = 10 muscles.