Activation of striated muscle implies that Ca2+ binding to troponin in the thin filament leads to a structural change in tropomyosin that makes the actin filament available for the interaction with the myosin motors. Biochemical and structural evidence suggests that the complete removal of the steric block of tropomyosin on the binding sites on actin depends on myosin motor attachment (McKillop and Geeves Biophys J 65:693, 1993). Myosin binding is likely responsible also for the spread of thin filament activation (Desai et al. J Biol Chem 290:1915, 2015), which determines the steepness of the sigmoidal force-pCa relation in skinned fibers (measured by the Hill coefficient nH), but the mechanism remains unclear. The question is investigated here by determining the force- and stiffness-pCa relations in skinned fibers from soleus muscle of rabbit (sarcomere length 2.4 μm), in which the force per motor is modulated by both temperature and addition of 1 μM Omecamtiv Mecarbil (OM). OM is known to (i) suppress force while remaining strongly bound to actin and (ii) increase Ca2+ sensitivity of thin filament shifting the force-pCa relation leftward while reducing its steepness (Caremani et al. Biophys J 114:644a, 2018; Woody et al. Nat Commun 9:3838, 2018). By using the half-sarcomere compliance analysis, we estimate the isometric force per myosin motor (F0), which ranges from 0.8 ± 0.1 pN (1 μM OM, 12°C) to 2.6 ± 0.1 pN (control, 25°C). Most importantly we find a linear relation between F0 and nH estimated from the force-pCa relations, whatever is the protocol used to modulate the two parameters. These results indicate that cooperativity in thin filament activation depends on the local stress induced by the attaching motor. Supported by ECRF and Telethon.

The contractile properties of muscle fibres have been extensively investigated by fast perturbation in sarcomere length to define the mechanical characteristics of myofilaments and myosin heads that underpin refined models of the acto-myosin cycle. Comparison of published data from intact fast-twitch fibres of frog muscle and demembranated fibres from fast muscle of rabbit shows that stiffness of the rabbit myosin head is only ∼62% of that in frog. To clarify if and how much the mechanical characteristics of the filaments and myosin heads vary in muscles of different animals we apply the same high resolution mechanical methods, in combination with X-ray diffraction, to fast-twitch fibres from the dogfish (Scyliorhinus canicula). The values of equivalent filament compliance (C(f)) measured by X-ray diffraction and in mechanical experiments are not significantly different; the best estimate from combining these values is 17.1 ± 1.0 nm MPa(−1). This value is larger than Cf in frog, 13.0 ± 0.4 nm MPa(−1). The longer thin filaments in dogfish account for only part of this difference. The average isometric force exerted by each attached myosin head at 5°C, 4.5 pN, and the maximum sliding distance accounted for by the myosin working stroke, 11 nm, are similar to those in frog, while the average myosin head stiffness of dogfish (1.98 ± 0.31 pN nm(−1)) is smaller than that of frog (2.78 ± 0.30 pN nm(−1)). Taken together these results indicate that the working stroke responsible for the generation of isometric force is a larger fraction of the total myosin head working stroke in the dogfish than in the frog.

In a contracting muscle, arrays of the dimeric motor protein myosin II pull the actin filament towards the centre of the sarcomere (the structural unit of the striated muscle) during cyclical ATP driven interactions. When the external load is smaller than the array force, the sarcomere works as a motor, converting metabolic energy into mechanical work; when the external load is larger than the array force, the sarcomere acts as a brake resisting the load with reduced metabolic cost. Due to the regular arrangement of molecular motors in striated muscle, X-ray diffraction is the best structural technique for imaging muscle contraction at molecular level. The high spacial resolution of the third generation synchrotrons (European Synchrotron Radiation Facility, ESRF, Grenoble, France and Advanced Photon Source, APS, Argonne, IL, USA) allows to record the fine structure of the meridional reflection originating from the 14.5 nm axial repeat of myosin motors, showing that the reflection is split in two peaks by the X-ray interference between the two arrays of myosin motors in each sarcomere [1]. Using the interference effect, the motion of myosin motors can be directly measured with subnanometer precision [2, 3].

Understanding of complex biological processes requires knowledge of molecular structures and measurement of their dynamics in vivo. The collective chemomechanical action of myosin molecules (the molecular motors) in the muscle sarcomere represents a paradigmatic example in this respect. Here, we describe a label-free imaging method sensitive to protein conformation in vivo. We employed the order-based contrast enhancement by second-harmonic generation (SHG) for the functional imaging of muscle cells. We found that SHG polarization anisotropy (SPA) measurements report on the structural state of the actomyosin motors, with significant sensitivity to the conformation of myosin. In fact, each physiological/biochemical state we probed (relaxed, rigor, isometric contraction) produced a distinct value of polarization anisotropy. Employing a full reconstruction of the contributing elementary SHG emitters in the actomyosin motor array at atomic scale, we provide a molecular interpretation of the SPA measurements in terms of myosin conformations. We applied this method to the discrimination between attached and detached myosin heads in an isometrically contracting intact fiber. Our observations indicate that isometrically contracting muscle sustains its tetanic force by steady-state commitment of 30% of myosin heads. Applying SPA and molecular structure modeling to the imaging of unstained living tissues provides the basis for a generation of imaging and diagnostic tools capable of probing molecular structures and dynamics in vivo.

The effects of absence of nebulin, an actin filament associated protein, on mechanical and kinetic properties of Ca2+-activated, chemically skinned, psoas fibers were investigated comparing mechanical performance of fibers from 1-day-old wildtype (wt) mice and 1-day-old nebulin deficient (nebulin−/−) mice. With fast mechanics (Linari et al. Biophys J 92:2476, 2007) on fiber bundles (sarcomere length 2.5μm, temperature 13 °C) we determined i) the relation between isometric force, stiffness and Ca2+ concentration; and ii) the unloaded shortening velocity and the power output at different loads at saturating Ca2+ (pCa, 4.50). Actin filament length in psoas fibers is not affected by the absence of nebulin, as proven by immunofluorescence imaging. Our results show a reduction in isometric force in the absence of nebulin without changes in the Ca2+ sensitivity of the contractile system. Stiffness measurements accompanied by analysis of the compliance of the half-sarcomere indicate that the reduction in isometric force is due to a proportional reduction in the number of myosin motors attached to actin without change in the average force of the motor. In addition, the absence of nebulin increases the unloaded shortening velocity by 63%, while decreases the maximum power output by 80%. These results indicate that the absence of nebulin induces a decrease of the rate of attachment of the myosin motors to actin and an increase of the rate of detachment of negatively strained motors under zero load, revealing a direct role for nebulin in stabilizing the actomyosin interaction. Supported by NIH and MiUR.

We use the inhibitor of isometric force of skeletal muscle N-benzyl-p-toluene sulfonamide (BTS) to decrease, in a dose dependent way, the number of myosin motors attached to actin during the steady isometric contraction of single fibers from frog skeletal muscle (4 degrees C, 2.1 mu m sarcomere length). In this way we can reduce the strain in the myofilament compliance during the isometric tetanus (T-o) from 3.54 nm in the control solution (T-o,T-NR) to -0.5 nm in 1 mu M BTS, where T-o is reduced to similar to 0.15 T-o,T-NR. The quick force recovery after a step release (1-3 nm per half-sarcomere) becomes faster with the increase of BTS concentration and the decrease of T-o. The simulation of quick force recovery with a multistate model of force generation, that adapts Huxley and Simmons model to account for both the high stiffness of the myosin motor (similar to 3 pN/nm) and the myofilament compliance, shows that the increase in the rate of quick force recovery by BTS is explained by the reduced strain in the myofilaments, consequent to the decrease in half-sarcomere force. The model estimates that i), for the same half-sarcomere release the state transition kinetics in the myosin motor are five times faster in the absence of filament compliance than in the control; and ii), the rate of force recovery from zero to T-o is similar to 6000/s in the absence of filament compliance.

Structural changes in myosin motors and filaments during relaxation from short tetanic contractions of intact single fibres of frog tibialis anterior muscles at sarcomere length 2.14 μm, 4°C were investigated by X‐ray diffraction. Force declined at a steady rate for several hundred milliseconds after the last stimulus, while sarcomere lengths remained almost constant. During this isometric phase of relaxation the intensities of the equatorial and meridional M3 X‐ray reflections associated with the radial and axial distributions of myosin motors also recovered at a steady rate towards their resting values, consistent with progressive net detachment of myosin motors from actin filaments. Stiffness measurements confirmed that the fraction of motors attached to actin declined at a constant rate, but also revealed a progressive increase in force per motor. The interference fine structure of the M3 reflection suggested that actin‐attached myosin motors are displaced towards the start of their working stroke during isometric relaxation. There was negligible recovery of the intensities of the meridional and layer‐line reflections associated with the quasi‐helical distribution of myosin motors in resting muscle during isometric relaxation, and the 1.5% increase in the axial periodicity of the myosin filament associated with muscle activation was not reversed. When force had decreased to roughly half its tetanus plateau value, the isometric phase of relaxation abruptly ended, and the ensuing chaotic relaxation had an exponential half‐time of ca 60 ms. Recovery of the equatorial X‐ray intensities was largely complete during chaotic relaxation, but the other X‐ray signals recovered more slowly than force.

Recently, the use of Second Harmonic Generation (SHG) for imaging biological samples has been explored with regard to intrinsic SHG in highly ordered biological samples. As shown by fractional extraction of proteins, myosin is the source of SHG signal in skeletal muscle. SHG is highly dependent on symmetries and provides selective information on the structural order and orientation of the emitting proteins and the dynamics of myosin molecules responsible for the mechano-chemical transduction during contraction. We characterise the polarization-dependence of SHG intensity in three different physiological states: resting, rigor and isometric tetanic contraction in a sarcomere length range between 2.0 mu m and 4.0 mu m. The orientation of motor domains of the myosin molecules is dependent on their physiological states and modulate the SHG signal. We can discriminate the orientation of the emitting dipoles in four different molecular conformations of myosin heads in intact fibers during isometric contraction, in resting and rigor. We estimate the contribution of the myosin motor domain to the total second order bulk susceptibility from its molecular structure and its functional conformation. We demonstrate that SHG is sensitive to the fraction of ordered myosin heads by disrupting the order of myosin heads in rigor with an ATP analog. We estimate the fraction of myosin motors generating the isometric force in the active muscle fiber from the dependence of the SHG modulation on the degree of overlap between actin and myosin filaments during an isometric contraction.

The design of planar arrays providing full azimuthal scanning coverage for skywave radar applications is considered in this work. The main objective is to simultaneously achieve a given beamwidth in any scanning direction and a given peak SLL in a specified subdomain of the visible region, while minimizing the number of required radiating elements. The capability of certain deterministic aperiodic arrays with substantially rotationally symmetric distributions of elements to comply with the full scan requirement while allowing the suppression of grating lobes is shown to be similar to that of random arrays.

The high degree of structural order in skeletal muscle allows imaging of this tissue by Second Harmonic Generation (SHG). As previously found (Vanzi et al, J. Muscle Cell Res. Motil. 2006) by fractional extraction of proteins, myosin is the source of SHG signal. A full characterization of the polarization-dependence of the SHG signal can provide very selective information on the orientation of the emitting proteins and their dynamics during contraction. We developed a line scan polarization method, allowing measurements of a full polarization curve in intact muscle fibers from skeletal muscle of the frog to characterize the SHG polarization dependence on different physiological states (resting, rigor and isometric tetanic contraction). The polarization data have been interpreted by means of a model in terms of the average orientation of SHG emitters. The different physiological states are characterized by distinct patterns of SHG polarization. The variation of the orientation of emitting molecules in relation to the physiological state of the muscle demonstrates that one part of SHG signal arises from the globular head of the myosin molecule that cross-links actin and myosin filaments. The dependence of the SHG modulation on the degree of overlap between actin and myosin filaments during an isometric contraction, provides the constraints to estimate the fraction of myosin heads generating the isometric force in the active muscle fiber.

The high degree of structural order in skeletal muscle allows imaging of this tissue by Second Harmonic Generation (SHG). Biochemical and colocalization studies have gathered an increasing wealth of clues for the attribution of the molecular origin of the muscle SHG signal to the motor protein myosin. Thus, SHG represents a potentially very powerful tool in the investigation of structural dynamics occurring in muscle during active production of force and/or shortening. A full characterization of the polarization-dependence of the SHG signal represents a very selective information on the orientation of the emitting proteins and their dynamics during contraction, provided that different physiological states of muscle (relaxed, rigor and active) exhibit distinct patterns of SHG polarization dependence. Here polarization data are obtained from single frog muscle fibers at rest and during isometric contraction and interpreted, by means of a model, in terms of an average orientation of the SHG emitters which are structured with a cylindrical symmetry about the fiber axis. The setup is optimized for accurate polarization measurements with SHG, combined with a line scan imaging method allowing acquisition of SHG polarization curves in different physiological states. We demonstrate that muscle fiber displays a measurable variation of the orientation of SHG emitters with the transition from rest to isometric contraction.

The intrinsically ordered arrays of proteins in skeletal muscle allows imaging of this tissue by Second Harmonic Generation (SHG). Biochemical and colocalization studies have gathered an increasing wealth of clues for the attribution of the molecular origin of the muscle SHG signal to the motor protein myosin. Thus, SHG represents a potentially very powerful tool in the investigation of structural dynamics occurring in muscle during active production of force. A full characterization of the polarization-dependence of the SHG signal represents a very selective information on the orientation of the emitting proteins and their dynamics during contraction, provided that different physiological states of muscle (relaxed, rigor and active) exhibit distinct patterns of SHG polarization dependence. Here polarization data are obtained from single frog muscle fibers at rest and during isometric contraction and interpreted, by means of a model, in terms of an average orientation of the SHG emitters which are structured with a cylindrical symmetry about the fiber axis. Optimizing the setup for accurate polarization measurements with SHG, we developed a line scan imaging method allowing measurement of SHG polarization curves in different physiological states. We demonstrate that muscle fiber displays a measurable variation of the orientation of SHG emitters with the transition from rest to isometric contraction.

Structural and mechanical changes occurring in the myosin filament and myosin head domains during the development of the isometric tetanus have been investigated in intact frog muscle fibres at 4°C and 2.15 μm sarcomere length, using sarcomere level mechanics and X‐ray diffraction at beamline ID2 of the European Synchrotron Radiation Facility (Grenoble, France). The time courses of changes in both the M3 and M6 myosin‐based reflections were recorded with 5 ms frames using the gas‐filled RAPID detector (MicroGap Technology). Following the end of the latent period (11 ms after the start of stimulation), force increases to the tetanus plateau value (T0) with a half‐time of 40 ms, and the spacings of the M3 and M6 reflections (SM3 and SM6) increase by 1.5% from their resting values, with time courses that lead that of force by ∼10 and ∼20 ms, respectively. These temporal relations are maintained when the increase of force is delayed by ∼10 ms by imposing, from 5 ms after the first stimulus, 50 nm (half‐sarcomere)−1 shortening at the velocity (V0) that maintains zero force. Shortening at V0 transiently reduces SM3 following the latent period and delays the subsequent increase in SM3, but only delays the SM6 increase without a transient decrease. Shortening at V0 imposed at the tetanus plateau causes an abrupt reduction of the intensity of the M3 reflection (IM3), whereas the intensity of the M6 reflection (IM6) is only slightly reduced. The changes in half‐sarcomere stiffness indicate that the isometric force at each time point is proportional to the number of myosin heads bound to actin. The different sensitivities of the intensity and spacing of the M3 and M6 reflections to the mechanical responses support the view that the M3 reflection in active muscle originates mainly from the myosin heads attached to the actin filament and the M6 reflection originates mainly from a fixed structure in the myosin filament signalling myosin filament length changes during the tetanus rise.

This review proposes a brief summary of two applications of lasers to muscle research. The first application (laser tweezers), is now a well-established technique in the field, adopted by several laboratories in the world and producing a constant stream of original data, fundamental for our improved understanding of muscle contraction at the level of detail that only single molecule measurements can provide. As an example of the power of this technique, here we focus on some recent results, revealing the performance of the working stroke in at least two distinct steps also in skeletal muscle myosin. A second laser-based technique described here is second-harmonic generation; the application of this technique to muscle research is very recent. We describe the main results obtained thus far in this area and the potentially remarkable impact that this technology may have in muscle research.

Abstract: Force and shortening in striated muscle are driven by a structural working stroke in the globular portion of the myosin molecules—the myosin head—that cross‐links the myosin‐containing filaments and the actin‐containing filaments. We use time‐resolved X‐ray diffraction in single fibers from frog skeletal muscle to link the conformational changes in the myosin head determined at atomic resolution in crystallographic studies with the kinetic and mechanical features of the molecular motor in the preserved sarcomeric structure. Our approach exploits the improved brightness and collimation of the X‐ray beams of the third generation synchrotrons by using X‐ray interference between the two arrays of myosin heads in each bipolar myosin filament to measure with Å sensitivity the axial motions of myosin heads in situ during the synchronous execution of the working stroke elicited by rapid decreases in length or load imposed during an active isometric contraction. Changes in the intensity and interference‐fine structure of the axial X‐ray reflections following the mechanical perturbation allowed to establish the average conformation of the myosin heads during the active isometric contraction and the extent of tilt during the elastic response and during the subsequent working stroke. The myosin working stroke is 12 nm at low loads, which is consistent with crystallographic studies, while it is smaller and slower at higher loads. The load dependence of the size and speed of the myosin working stroke is the molecular determinant of the macroscopic performance and efficiency of muscle.

X‐ray diffraction patterns were recorded from isolated single fibres of frog skeletal muscle during isometric contraction at temperatures between 0 and 17°C. Isometric force was 43 ± 2% (mean ±s.e.m., n= 10) higher at 17°C than 0°C. The intensity of the first actin layer line increased by 57 ± 18% (n= 5), and the ratio of the intensities of the equatorial 1,1 and 1,0 reflections by 20 ± 7% (n= 10), signalling radial or azimuthal motions of the myosin head domains. The M3 X‐ray reflection from the axial repeat of the heads along the filaments was 27 ± 4% more intense at 17°C, suggesting that the heads became more perpendicular to the filaments. The ratio of the intensities of the higher and lower angle peaks of the M3 reflection (RM3) was 0.93 ± 0.02 (n= 5) at 0°C and 0.77 ± 0.02 at 17°C. These peaks are due to interference between the two halves of each myosin filament, and the RM3 decrease shows that heads move towards the midpoint of the myosin filament at the higher temperature. Calculations based on a crystallographic model of the heads indicated that the observed RM3 change corresponds to tilting of their light‐chain domains by 9 deg, producing an axial displacement of 1.4 nm, which is equal to that required to strain the actin and myosin filaments under the increased force. We conclude that the higher force generated by skeletal muscle at higher temperature can be accounted for by axial tilting of the myosin heads.