The Synergic Role of Actomyosin Architecture and Biased Detachment in Muscle Energetics: Insights in Cross Bridge Mechanism beyond the Lever-Arm Swing

Figure 1. Cross-bridge cycle and pseudo-replication of the 3D structure in the model. (A) The cross-bridge cycle where, in an otherwise classical model, the super-relaxed state in myosin motors is taken into account, splitting the whole cycle into a resting cycle for the activation of the thick filament and a working cycle, where a two-step power stroke generates force. The activation rate depends on the force acting on the thick filament (see Equation (6)). The attachment and detachment rates depend on the relative position of the motor with respect to its anchor (Equations (7) and (8)). (B) 3D structure of one thick filament surrounded by six thin filaments; 4 crowns of motors are shown. The two helixes on the thin filaments are indicated along with the available binding regions, highlighted with the color of the corresponding motor heads. (C) Lateral view of the 3D structure. Only the binding regions for the deep red motors are indicated, showing La/3 shifts (see Section 4.1). The mismatch between actin-binding regions and the motors can be particularly appreciated in the motors pointing out of the figure plane toward the viewer (deep red dots in the middle of the figure) and the…

Figure 2. Comparison of the uniform binding region model vs. the limited target zones model, which generates geometrical hindrance. (A) The attachment rate kdON−a0 is adjusted to fit the F(v) curve (experimental data for Rana temporaria from [9], velocity in µm per second per half sarcomere (hs)). Then, the detachment rate kax−dON is adjusted to have 30% of motors attached in isometric contraction (experimental data for Rana temporaria from [9]). (B) The rate of activation of the thick filament as a function of the force kdOFF−dON(Fj) is adjusted to fit the kinetics of the force generation during activation and force recovery after an unloaded shortening (experimental data for Rana temporaria from [12]). (C) Despite the mechanical behaviors being very similar for the two models, the uniformly distributed target zones (purple circles) induce a high rATPiso, while the limited target zones drastically reduce it (black triangles).

Figure 3. Analysis of the effect of biased kinetics and geometrical hindrance on rATP(v). Black triangles refer to the model with limited target zones and biased kinetics for comparison. (A) Both the limited target zones and the preferential detachment are excluded (orange squares): the ATP-ase rate does not depend on the velocity of shortening except at low external forces (high velocity) due to the mechano-sensing mechanism. When the limited target zone is present without the effect of the preferential detachment (yellow circles), the rATPmax/rATPiso ratio is lower than the experimental value. (B) The geometrical hindrance is included without biased attachment (purple circles) or without biased detachment (blue squares). The high rATPmax/rATPiso ratio is maintained only when the geometrical hindrance acts together with the preferential detachment of myosin motors. On the contrary, the preferential attachment has no effect on the rATP(v) relationship.

Figure 4. In vitro actin sliding assay driven by a DNA rod-myosin complex. (A) Schematic of the in vitro sliding assay. Qdots (QDs) were attached to an actin filament, and a DNA rod-myosin complex was adsorbed onto a glass slide. Biotins were attached to the bottom of the DNA rod to fix the rod to the neutravidin-coated glass slide, and DNA handles (21-base single-stranded DNA) were attached to the top of the DNA rod (see cross section shown in right cartoon) at 14.3 nm spacing along the DNA rod. Because anti-handles were labeled to myosins, the myosins were precisely positioned to the DNA rods with a spacing of 14.3 nm. ATP concentration, 0.5 µM. (B) Trajectories of the actin sliding distance for three distinct myosin mutants. Purple—no light chains (without lever-arm); green—WT; blue—four light chains (double-length lever-arm). (C) Predicted average velocities of the sliding model (triangles) at different step sizes and the corresponding velocities observed experimentally (dots, colors as in panel B) are related to different lengths of the lever-arm. The intercept at zero step-size is predicted by the model preventing the motor to swing the lever-arm. Experimental data were obtained from…