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Force generation by skeletal muscle is controlled by mechanosensing in myosin filaments

Abstract

Contraction of both skeletal muscle and the heart is thought to be controlled by a calcium-dependent structural change in the actin-containing thin filaments, which permits the binding of myosin motors from the neighbouring thick filaments to drive filament sliding1,2,3. Here we show by synchrotron small-angle X-ray diffraction of frog (Rana temporaria) single skeletal muscle cells that, although the well-known thin-filament mechanism is sufficient for regulation of muscle shortening against low load, force generation against high load requires a second permissive step linked to a change in the structure of the thick filament. The resting (switched ‘OFF’) structure of the thick filament is characterized by helical tracks of myosin motors on the filament surface and a short backbone periodicity2,4,5. This OFF structure is almost completely preserved during low-load shortening, which is driven by a small fraction of constitutively active (switched ‘ON’) myosin motors outside thick-filament control. At higher load, these motors generate sufficient thick-filament stress to trigger the transition to its long-periodicity ON structure, unlocking the major population of motors required for high-load contraction. This concept of the thick filament as a regulatory mechanosensor provides a novel explanation for the dynamic and energetic properties of skeletal muscle. A similar mechanism probably operates in the heart.

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Figure 1: X-ray reflections associated with thick-filament structure in isolated skeletal muscle fibres depend on the external load during activation.
Figure 2: The OFF structure of the thick filament is transiently restored when the load on a fully active muscle fibre is removed.
Figure 3: The time course of force development is controlled by the regulatory state of the thick filament.
Figure 4: Dual-filament regulation in skeletal muscle.

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Acknowledgements

We thank M. Dolfi and J. Gorini for electronic and mechanical engineering support and P. Panine for assistance at the beamline. We thank ESRF for beamtime, and Ente Cassa di Risparmio di Firenze 2010.1402, FIRB-Futuro in Ricerca project RBFR08JAMZ, MIUR-PRIN project 2010R8JK2X (Italy), MRC (UK) and ESRF for financial support.

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Authors and Affiliations

Authors

Contributions

M.L., E.B., M.R., L.F., M.C., G.P., V.L. and M.I. contributed to the conception and design of the experiments, the collection, analysis and interpretation of data, and drafting or critical revision of the article. T.N. contributed to data collection and analysis.

Corresponding author

Correspondence to Vincenzo Lombardi.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 The increase in the spacing of the M6 reflection on activation is delayed by imposing a period of unloaded shortening.

The top traces show SM6 (circles) superimposed on force (continuous line); filled/open circles and thicker/thinner line denote data from fixed-end tetani and tetani with imposed shortening, respectively. The bottom traces show imposed length change (ΔL, expressed as percentage of initial fibre length (% L0)). X-ray data added from one/two repeats of the protocol in three muscle fibres.

Source data

Extended Data Figure 2 Stiffness changes during unloaded shortening.

a, Half-sarcomere stiffness (e) relative to that at the plateau of an isometric tetanus (e0), at different times after the start of unloaded shortening of 10% L0 applied at the tetanus plateau, calculated from the ratio of force and half-sarcomere length changes in response to 0.2% L0 step stretches complete in 100 μs. Mean ± s.e.m. from four fibres; species, Rana esculenta, 4 °C; e0 = 0.27 ± 0.01 T0 nm−1; T0 = 137 ± 13 kPa. b, Fraction of myosin motors attached to actin (fA; thick solid line) and fractions with bound ATP (thin solid line) or ADP and inorganic phosphate (Pi) (dashed line) as a function of time during unloaded shortening, calculated from the kinetic model described in the Supplementary Discussion. c, Number of myosin motors attached to actin (n) relative to that at the plateau of an isometric tetanus (n0), at different times during unloaded shortening, calculated from e/e0 in a as described in the Supplementary Discussion. Mean ± s.e.m. from four fibres; s.e.m. includes the contribution of errors in measured values of filament and parallel elasticity. The thick line was calculated by normalizing fA in b for its tetanus plateau value.

Source data

Extended Data Figure 3 Changes in the intensity, spacing and interference fine structure of the M3 reflection associated with a period of unloaded shortening.

Top, sarcomere length change in nanometres per half-sarcomere. ac, Changes in intensity (IM3; a), spacing (SM3; b) and interference fine structure (LM3; c) (circles) superimposed on force (continuous line). Horizontal dashed lines, resting value of X-ray parameter; vertical dashed lines, time of end of shortening. X-ray data added from three fibres. Filled/open circles denote data before/after the end of shortening.

Source data

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion and additional references. (PDF 295 kb)

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Linari, M., Brunello, E., Reconditi, M. et al. Force generation by skeletal muscle is controlled by mechanosensing in myosin filaments. Nature 528, 276–279 (2015). https://doi.org/10.1038/nature15727

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