Mechanical parameters of the molecular motor myosin II determined in permeabilised fibres from slow and fast skeletal muscles of the rabbit

Abstract

Key points: The different performance of slow and fast muscles is mainly attributed to diversity of the myosin heavy chain (MHC) isoform expressed within them. In this study fast sarcomere-level mechanics has been applied to Ca²⁺ -activated single permeabilised fibres isolated from soleus (containing the slow myosin isoform) and psoas (containing the fast myosin isoform) muscles of rabbit for a comparative definition of the mechano-kinetics of force generation by slow and fast myosin isoforms in situ. The stiffness and the force of the slow myosin isoform are three times smaller than those of the fast isoform, suggesting that the stiffness of the myosin motor is a determinant of the isoform-dependent functional diversity between skeletal muscles. These results open the question of the mechanism that can reconcile the reduced performance of the slow MHC with the higher efficiency of the slow muscle.

Abstract: The skeletal muscle exhibits large functional differences depending on the myosin heavy chain (MHC) isoform expressed in its molecular motor, myosin II. The differences in the mechanical features of force generation by myosin isoforms were investigated in situ by using fast sarcomere-level mechanical methods in permeabilised fibres (sarcomere length 2.4 μm, temperature 12°C, 4% dextran T-500) from slow (soleus, containing the MHC-1 isoform) and fast (psoas, containing the MHC-2X isoform) skeletal muscle of the rabbit. The stiffness of the half-sarcomere was determined at the plateau of Ca²⁺ -activated isometric contractions and in rigor and analysed with a model that accounted for the filament compliance to estimate the stiffness of the myosin motor (ε). ε was 0.56 ± 0.04 and 1.70 ± 0.37 pN nm^-1 for the slow and fast isoform, respectively, while the average strain per attached motor (s₀ ) was similar (∼3.3 nm) in both isoforms. Consequently the force per motor (F₀ = εs₀ ) was three times smaller in the slow isoform than in the fast isoform (1.89 ± 0.43 versus 5.35 ± 1.51 pN). The fraction of actin-attached motors responsible for maximum isometric force at saturating Ca²⁺ (T_0,4.5 ) was 0.47 ± 0.09 in soleus fibres, 70% larger than that in psoas fibres (0.29 ± 0.08), so that F₀ in slow fibres was decreased by only 53%. The lower stiffness and force of the slow myosin isoform open the question of the molecular basis of the higher efficiency of slow muscle with respect to fast muscle.

Keywords: fast myosin isoform; muscle mechanics; myosin motor force; myosin motor stiffness; slow myosin isoform.

Figure 1. Intermyofibrillar space quantification

A–F, representative electron micrographs of cross sections of fibres from soleus (upper row) and psoas (lower row) muscles fixed before (A and D) after the skinning procedure (B and E) and after the skinning procedure in the presence of 4% dextran (C and F). The intermyofibrillar space is labelled in cyan. Scale bar: 0.5 μm. The percentage of myofibrillar area and intermyofibrillar space area over the total area are reported in the table for the slow and fast fibres as means ± SEM. Student's t test: ^* P < 0.05 with respect to the intact value within each fibre type.

Figure 2. Force development following a T‐jump in slow (A) and fast (B) fibres and identification of fibre types (C)

A and B, upper panels, hs length change; lower panels, force response. The horizontal line in the lower panels indicates zero force. During the period (a) the fibre travels in air and within the shadow zone of the chamber. The dashed vertical lines mark the start of the period (b) when the striation follower signal is recorded. Slow fibre (A): fibre length, 5 mm; segment length under the striation follower, 1.1 mm; average segment sarcomere length, 2.48 μm; test temperature, 12.5°C; CSA 5000 μm². Fast fibre (B): fibre length, 5.2 mm; segment length under the striation follower, 0.9 mm; average segment sarcomere length, 2.46 μm; test temperature, 12.4°C; CSA, 5700 μm². C, MHC isoform identification in the fibre by SDS‐PAGE in the area of migration of myosin heavy chain (upper panel) and actin (lower panel). Lane a: single fibre from soleus muscle, which shows pure MHC‐1content; lane b: single fibre from psoas muscle, which shows pure MHC‐2X content. On the right of the lanes are shown the projections of the mass density along the vertical axis after horizontal integration: grey, slow fibre; black, fast fibre. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 3. Protocol for measuring the half‐sarcomere stiffness of a slow fibre during isometric contraction at saturating [Ca²⁺] (A) and at a similar force in rigor (B)

Upper trace, length change per half‐sarcomere; lower trace, force. The same step sequence was imposed on both activated and rigor fibre. Step amplitudes in the sequence were −1.5, +1.5, −3.0, +3.0 nm per hs. A step of opposite direction was imposed 50 ms after each test step to return the length and the force close to their values before the step. The interval between the test steps was 200 ms. Force is expressed relative to the CSA in relaxing solution (5150 μm²). Fibre length, 5.3 mm; segment length under the striation follower, 0.93 mm; average segment sarcomere length, 2.45 μm; test temperature, 12.2°C. For rigor (same fibre), average segment sarcomere length, 2.42 μm; temperature, 12.1°C; CSA, 4030 μm².

Figure 4. Measurements of the half‐sarcomere stiffness during isometric contraction and in rigor

A and C, superimposed hs length changes (upper traces) and force response (lower traces) for steps of different sizes in activating solution at saturating [Ca²⁺] (left column) and in rigor (right column) at a force about T _0,4.5 in a slow fibre (A) and in a fast fibre (C). B and D, T ₁ relations at different pCa (open symbols) and in rigor at forces about T _0,4.5 and 1/3 × T _0,4.5 (filled symbols) determined in a slow fibre (B) and in a fast fibre (D). Lines are linear regression equations fit to the active (dashed) or rigor (continuous) data. The abscissa intercept of each line is the average strain of the half‐sarcomere before the length step (Y ₀). Slow fibre: same fibre as in Fig. 3. Fast fibre: fibre length, 5.3 mm; segment length under the striation follower, 0.93 mm; average segment sarcomere length, 2.46 μm; test temperature, 12.2°C; CSA, 5700 μm².

Figure 5. Half‐sarcomere stiffness in isometric contraction and in rigor and derived parameters

In all panels filled circles and continuous lines refer to slow fibres and open circles and dashed lines to fast fibres. Dependence on force of hs‐stiffness (A), hs‐compliance (B) and hs‐strain (C). The lines in A connect the average T _0,4.5 (larger symbols) with the origin. In C the lines are the linear regression fit to data for forces >40 kPa. In B the lines are calculated with the parameters estimated from the fits in C. D, hs–strain relation in rigor; the lines are the linear regressions fit to data forced to pass through zero. Data from 9 and 10 fibres for soleus and psoas, respectively.

Figure 6. Models representing the elements contributing to the half‐sarcomere compliance (A and B), and dependence of the hs compliance on force in slow (C) and fast (D) fibres

In Model 1 (A), the myofilament compliance (C _f) is in series with an array of myosin motor with a compliance s/T. In Model 2 (B) an elastic element with compliance C _P is in parallel with the array of motors. In C and D experimental data are the same as those reported in Fig. 5 B; lines represent the C _hs–force relations calculated in the absence (black line, eqn (1) from Model 1) or in the presence (grey line, eqn (3) from Model 2) of C _P, the elastic element in parallel with the myosin motors. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 7. Amino acid sequences of the converter domain of MHC‐1 and MHC‐2X isoforms in six species of mammals

Numbers at the beginning and at the end of each sequence are the residues of start and end of the domain. Differences between slow and fast isoforms within a species are in bold. Myosin heavy chain sequences were found at the NCBI (National Centre for Biotechnological Information; Bethesda, MD, USA). The sequences of the converter region were aligned using the BLAST program available at NCBI.