102 103 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 Controlling the strength ofmuscles Related publication: Hill, C., Brunello, E., Fusi, L., Ovejero, J. G., & Irving, M. Myosin-based regulation of twitch and tetanic contractions in mammalian skeletal muscle. ELife , 10, (2021). DOI: 10.7554/eLife.68211 Publication keywords: Skeletal muscle; Muscle regulation; Muscle contraction; X-ray diffraction S keletal muscles connect to bones and allow a wide range of movements and functions, and muscle weakness caused by disease and ageing significantly impacts quality of life. Although these changes are generally associated with loss of muscle mass, the intrinsic strength of a givenmass of muscle is also reduced. The basicmechanical response of a skeletal muscle cell to the electrical stimulus it receives fromthe central nervous system is called a twitch. The force produced by a twitch ismuch smaller than themaximum force that themuscle cell can producewhen themotor proteins that drive contraction are fully activated. As themotor proteins inmuscles are organised into regular (almost crystalline) arrays, the structural changes that control muscle activation can be followed by measuring the diffraction of X-rays by the motor arrays. However, muscles diffract X-rays very weakly, and these experiments require an extremely bright X-ray source combined with a sensitive X-ray video camera that can record 200 diffraction patterns every second. The Small Angle Scattering and Diffraction beamline (I22) at Diamond Light Source is one of few facilities worldwide thatmeet these demanding requirements. The discovery of the molecular structural factors that limit the strength of skeletal muscle in its normal twitch response allows those structures to be used to design and test potential drugs that could increase muscle strength. Almost identical mechanisms operate to limit the strength of the heartbeat and could be targeted to treat heart failure. Muscle contraction is driven by sliding between two types of protein polymer filaments – the actin and myosin filaments. The former acts as a track for myosin motors to ‘walk’ along, stepping between actin monomers. In resting muscle, the actin monomers are not available for myosin motors to bind because regulatory proteins in the actin filaments block the binding sites. Activation of the muscle by an electrical signal from the central nervous system removes that block through a well-characterised signalling pathway mediated by a transient increase in the calcium concentration in the muscle cell. However, there is a second block on the interaction between myosin and actin in resting muscle: nearly all the myosin motors are locked into helical tracks on the surface of the myosin filaments (Fig. 1a; yellow motors), producing a characteristic‘layer line’reflection in the X-ray pattern from restingmuscle (Fig. 1c, ‘ML1’). When the muscle contracts the motors leave the helical tracks and bind to actin (Fig. 1b; purple motors), producing a brighter reflection on the vertical axis of the pattern (Fig. 1d, ‘M3’). These characteristic X-ray reflections were used to determine how the strength and speed of the muscle response to a single electrical stimulus from the brain is controlled. It was known that this force response - called a twitch (Fig. 2a; blue) - is much smaller than the maximum force the muscle can produce when activated repetitively by a train of stimuli, called a tetanus (Fig 2a; navy). However, it was also known that the calcium transient in a twitch is large enough to saturate all the regulatory sites to which it binds in the actin filaments. This suggests that the size and speed of the twitch might be limited by the fraction of myosin motors that leave the helical tracks. To test that idea, X-ray patterns like those in Fig. 1c,d were recorded every five milliseconds during twitch and tetanic contractions using a Pilatus 2Mdetector at the I22 beamline 1 . The brightness of the ML1 reflection, shown in Fig. 2b as its amplitude (AML1), which is proportional to the fraction of myosin motors in the helical array (yellow motors in Fig. 1a), decreased rapidly at the start of contraction, in both a twitch (blue) and a tetanus (navy). However, the decrease in the twitch was rapidly reversed, so that it never became as large as in a tetanus. These results, supported by similar analyses of the changes in other parts of the diffraction patterns, were used to create a structural model of the movements of the myosin motors during a twitch and a tetanus (Fig. 3). In resting muscle (Fig. 3, lower panel 1) nearly all the myosin motors are locked into helical tracks, and both filaments can be considered to be switched off, coded in the diagram as a white filament backbone. At the peak response to a tetanus (Fig. 3, navy), both the actin and myosin filaments are maximally switched on, coded by the red and green filament backbones respectively (panel 3), although some motors remain in the helical tracks. At the peak of the twitch (blue, panel 2), the actin filament is fully on (red), but the myosin filament is only partly activated (light green); a large fraction of the myosin motors stay in their helical tracks. Thus, the strength of muscle in its unitary response to electrical stimulation- the twitch- is limited by the fraction of myosin motors that are activated. The speed of force development is limited by the rate of motors binding to the actin filaments rather than by the speed of actin filament activation, and the speed of relaxation is determined by the rate of detachment of the motors, which is againmuch slower than the inactivation of the actin filaments. These results challenge the previous focus on the actin filaments and the calcium transient as targets for therapeutic intervention in muscle weakness and heart disease, suggesting that the myosin filament may be a more functionally relevant target. The I22 beamline with a Pilatus 2M detector is ideally suited to build on these results to exploit that therapeutic strategy. The next steps in the programme would be to investigate the structural mechanisms of myosin filament activation and relaxation, of the adaptation of the myosin motors to the variable external loads that muscles experience in the body, and of the enhanced response of muscles that have been recently stimulated, called post-tetanic potentiation. References: 1. Hill, C. et al. Myosin-based regulation of twitch and tetanic contractions in mammalian skeletal muscle. ELife , 10 (2021). DOI: 10.7554/ eLife.68211 Funding acknowledgement: Diamond Light Source beamtime at I22 was granted on project SM21316- 1. This work was funded by the Medical Research Council MR/R01700X/1 and Diamond Light Source. EB and JGO were supported by a British Heart Foundation Intermediate Basic Science Research Fellowship awarded to EB (FS/17/3/32604). LF was funded by a Sir Henry Dale Fellowship awarded by theWellcome Trust and the Royal Society (210464/Z/18/Z). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Corresponding author: Dr Cameron Hill, King’s College London,
[email protected] Soft CondensedMatter Group Beamline I22 Figure 1: Schematic of the myosin and actin filaments and X-ray diffraction patterns recorded at rest ( a & c ) and in a tetanus ( b , d ); The helical tracks of myosin motors (yellow) ( a ) generate the first-order myosin layer line reflection (ML1) ( c, red box); In the tetanus ( b, d ), some actin-attached myosin motors (purple) and non-actin-attached partner myosin motors (magenta), intensify the M3 reflection ( d , green box); Isotropic motors (light pink) do not contribute to the diffraction patterns. Figure 2: ( a ) Time-course of isometric force production relative to maximal tetanic force (T/T 0 ) for two stimulation protocols (black trace) in response to a single stimulus (twitch; blue) or a 100ms train of electrical stimuli (tetanus; navy); ( b ) Time-course of the amplitude of the ML1 reflection (A ML1 ) normalised to the mean resting value for the twitch (open, blue) and tetanus (filled, navy). Mean ± SEM for n=5 muscles for tetanus and n=4 muscles for twitch. Figure 3: Motor conformations and activation level of the myosin (green cylinder) and actin (red cylinder) filaments for the twitch (blue) and tetanus (navy); Panel 1, rest; panel 2, peak twitch force; panel 3, peak tetanus force. The darker the colour of the filament, the higher its activation level.