Electrostatic interaction of loop 1 and backbone of human cardiac myosin regulates the rate of ATP induced actomyosin dissociation

Double mutation D208Q:K450L was introduced in the beta isoform of human cardiac myosin to remove the salt bridge D208:K450 connecting loop 1 and the seven stranded beta sheet within the myosin head. Beta isoform specific salt bridge D208:K450 was previously discovered in the molecular dynamics simulations. It was proposed that loop 1 modulates nucleotide affinity to actomyosin and we hypothesized that the electrostatic interactions between loop 1 and myosin head backbone regulates ATP binding to and ADP dissociation from actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutant of the myosin head construct (843 amino acid residues) were expressed in differentiated C2C12 cells, and the kinetics of ATP induced actomyosin dissociation and ADP release were characterized using transient kinetics spectrophotometry. Both constructs exhibit a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. We observed a faster rate of ATP induced actomyosin dissociation with the mutant, compared to the wild type actomyosin. The rate of ADP release from actomyosin remains the same for the mutant and the wild type actomyosin. We conclude that the flexibility of loop 1 is a factor affecting the rate of ATP binding to actomyosin and actomyosin dissociation. We observed no effect of loop 1 flexibility on the rate of ADP release from actomyosin.

S2 domain gives myosin filaments some flexibility

JGP microscopy study supports the idea that the region linking myosin head and tail domains can be peeled away from filament backbone to prevent actin-attached heads from impeding filament movement.

The N terminus of myosin-binding protein C extends toward actin filaments in intact cardiac muscle

Myosin and actin filaments are highly organized within muscle sarcomeres. Myosin-binding protein C (MyBP-C) is a flexible, rod-like protein located within the C-zone of the sarcomere. The C-terminal domain of MyBP-C is tethered to the myosin filament backbone, and the N-terminal domains are postulated to interact with actin and/or the myosin head to modulate filament sliding. To define where the N-terminal domains of MyBP-C are localized in the sarcomere of active and relaxed mouse myocardium, the relative positions of the N terminus of MyBP-C and actin were imaged in fixed muscle samples using super-resolution fluorescence microscopy. The resolution of the imaging was enhanced by particle averaging. The images demonstrate that the position of the N terminus of MyBP-C is biased toward the actin filaments in both active and relaxed muscle preparations. Comparison of the experimental images with images generated in silico, accounting for known binding partner interactions, suggests that the N-terminal domains of MyBP-C may bind to actin and possibly the myosin head but only when the myosin head is in the proximity of an actin filament. These physiologically relevant images help define the molecular mechanism by which the N-terminal domains of MyBP-C may search for, and capture, molecular binding partners to tune cardiac contractility.

Muscle contraction as a Markov Process - II: x-ray interference data (M3 & M6 reflections) imply myosin cross-bridge motions are controlled by structural transitions along actin filaments

The unit underlying the construction and functioning of muscle fibers is the sarcomere. Tension develops in fibers as thousands of sarcomeres arranged in series contract in unison. Shortening is due to the sliding of actin thin filaments along antiparallel arrays of myosin thick filaments. Remarkably, myosin catalytic heads situated across the center M-line of a sarcomere are separated by a distance that is a half integral of the 14.5 nm spacing between successive layers of myosin heads on the thick filaments. This results in the splitting of the 14.5 nm meridional reflection in X-ray diffraction patterns of muscle fibers. Following a quick drop in tension, changes in the relative intensities of the split meridional peaks provide a sensitive measure of myosin head movements. We use published data obtained with the x-ray interference method to validate a theory of muscle contraction in which cooperative structural transitions along force-generating actin filaments regulate the binding of myosin heads. The probability that an actin-bound myosin head will detach is represented here by a statistical function that yields a length-tension curve consistent with classical descriptions of the recovery of contracting muscle fibers subjected to millisecond drops in tension.

Abstract 448: Strain Rate Dependent Myosin Head Position Changes During Relaxation

Impaired cardiac relaxation is present in nearly all cases of heart failure and possibly in up to 25% of the asymptomatic population. Myocardial relaxation is known to be biochemically modified by the calcium reuptake rate, thin filament calcium sensitivity, and crossbridge kinetics. Mechanical regulation of relaxation was thought to be regulated via afterload, but we have recently shown that a lengthening strain was sufficient to modify relaxation. Further, the relaxation rate is actually dependent on the strain rate, a relationship that we termed Mechanical Control of Relaxation. Computational modeling suggests that myosin detachment is a key mechanism underlying Mechanical Control of Regulation, but to date, no experimental evidence for this was available. The objective of this study was to determine if myosin head position changed in response to lengthening strains during relaxation. Intact cardiac trabeculae were mounted within the beamline of the Biophysical Collaborative Access Team (BioCAT) beamline at the Advanced Photon Source at Argonne National Laboratories. The trabeculae were paced and load-clamps were performed during time-resolved imaging of the equatorial axis, which primarily reflects myosin head positioning. Activation (pacing) caused the myosin head localization to shift from the thick filament to near the thin filament (increased I 1,1 /I 1,0 ratio). During stretch, there was a transient decline of the I 1,1 /I 1,0 ratio which recovered until relaxation was complete, when the ratio again reduced indicating myosin returned to the thick filament. These preliminary data suggest that Mechanical Control of Relaxation is caused by perturbations in myosin, but the late-diastolic kinetics suggests that the strain-rate dependent detachment does not lead to immediate deactivation of myosin heads. Modifications of myosin ATPase properties may reveal more specific regulatory targets, which may provide new insight and targets for treating impaired myocardial relaxation.

The time of strong actomyosin binding depends on electrostatic interactions within the force generating region in human cardiac myosin

Two single mutations, R694N and E45Q, were introduced in the beta isoform of human cardiac myosin to remove permanent salt bridges E45:R694 and E98:R694 in the force-generating region of myosin head. Beta isoform-specific bridges E45:R694 and E98:R694 were discovered in the molecular dynamics simulations of the alpha and beta myosin isoforms. Alpha and beta isoforms exhibit different kinetics, ADP dissociates slower from actomyosin containing beta myosin isoform, therefore, beta myosin stays strongly bound to actin longer. We hypothesize that the electrostatic interactions in the force-generating region modulate affinity of ADP to actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutants of the myosin head construct (1-843 amino acid residues) were expressed in differentiated C2C12 cells, and duration of the strongly bound state of actomyosin was characterized using transient kinetics spectrophotometry. All myosin constructs exhibited a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. Mutant R694N showed faster rate of ADP release from actomyosin, compared to the wild type and the E45Q mutant, thus confirming that electrostatic interactions within the force-generating region of human cardiac myosin regulate ADP release and the duration of the strongly bound state of actomyosin.

X-ray Diffraction Studies on the Structural Origin of Dynamic Tension Recovery Following Ramp-Shaped Releases in High-Ca Rigor Muscle Fibers

It is generally believed that during muscle contraction, myosin heads (M) extending from myosin filament attaches to actin filaments (A) to perform power stroke, associated with the reaction, A-M-ADP-Pi → A-M + ADP + Pi, so that myosin heads pass through the state of A-M, i.e., rigor A-M complex. We have, however, recently found that: (1) an antibody to myosin head, completely covering actin-binding sites in myosin head, has no effect on Ca2+-activated tension in skinned muscle fibers; (2) skinned fibers exhibit distinct tension recovery following ramp-shaped releases (amplitude, 0.5% of Lo; complete in 5 ms); and (3) EDTA, chelating Mg ions, eliminate the tension recovery in low-Ca rigor fibers but not in high-Ca rigor fibers. These results suggest that A-M-ADP myosin heads in high-Ca rigor fibers have dynamic properties to produce the tension recovery following ramp-shaped releases, and that myosin heads do not pass through rigor A-M complex configuration during muscle contraction. To obtain information about the structural changes in A-M-ADP myosin heads during the tension recovery, we performed X-ray diffraction studies on high-Ca rigor skinned fibers subjected to ramp-shaped releases. X-ray diffraction patterns of the fibers were recorded before and after application of ramp-shaped releases. The results obtained indicate that during the initial drop in rigor tension coincident with the applied release, rigor myosin heads take up applied displacement by tilting from oblique to perpendicular configuration to myofilaments, and after the release myosin heads appear to rotate around the helical structure of actin filaments to produce the tension recovery.