Studies of the Super-Relaxed State and Interacting-Heads Motif in Beta-Cardiac Myosin

Abstract

The interacting-heads motif (IHM) is a structure of myosin that has been proposed to modulate cardiac output by occluding myosin molecules from undergoing the force-generating cycle. It is hypothesized to be the structural basis for the super-relaxed state (SRX), a low-ATPase kinetic state thought to be cardioprotective. The IHM was first seen in EM images of myosin filaments, and we have now measured it in isolated myosin molecules. The SRX was first seen in permeabilized muscle fibers, and we can now measure it in isolated myosin molecules as well. The goal of this work was to test this hypothesis by determining directly and quantitatively the fractions of myosin in the IHM and SRX under the same conditions in solution. To detect the structural IHM, we used time-resolved fluorescence resonance energy transfer (TR-FRET).We label opposite regulatory light chain (RLC), one of two light chains attached to the main heavy chain molecule, on myosin molecules in a variety of myosin preparations. Using a truncated version of myosin, we find that the TR-FRET labeling scheme can observe two distinct populations. One population was observed at a center distance of 2.0 nm, whereas the other was not detectable by FRET, implying a distance greater than 4 nm. We identified the 2.0 nm population as the IHM by applying the same crosslinking protocol used previously to image the IHM by electron microscopy. Under the same conditions, we also measured the fraction of myosin in the SRX using stopped-flow kinetics. Our results show that the populations of SRX and IHM myosin were similar, unless treated with mavacamten, a drug that recently completed Phase-III clinical trials to treat hypertrophic cardiomyopathy (HCM) and is proposed to act by stabilizing both the SRX and IHM. However, we found that mavacamten had a much greater effect on the SRX (55% increase) than on the IHM (4% increase). We conclude that the IHM structure is sufficient but not necessary to produce the SRX kinetic state. We found that, interestingly, the time-resolved FRET results of the thick filaments better fit to two populations instead of the single FRET population of the isolated myosin molecules. One population was at a similar distance to that seen in isolated myosin molecules, and a new distinct population at a longer distance between the fluorophores. It is likely that this new population is the open myosin conformation, because of its longer distance and wider distribution. Native thick filaments (nTFs) were found to have more FRET than synthetic thick filaments (sTFs). That is consistent with the hypothesis that the accessory proteins in the nTFs are important in stabilizing the IHM. In conclusion, the IHM was observed in different myosin preparations, allowing for more studies to probe the relationship between the IHM and heart contractility.