Observing the Myosin Super-Relaxed State (SRX) in Cardiac Thick Filaments

Abstract

We aim to develop time-resolved fluorescence resonance energy transfer (FRET) sensors to detect the structural state of myosin in cardiac thick filaments. In relaxation, these filaments are hypothesized to isomerize between the disordered relaxed state (DRX), which has the potential to interact with actin and generate force in the sarcomere, and an inactive state termed super-relaxed (SRX), which has much lower ATPase activity than DRX, apparently due to inhibitory interactions between the two heads of myosin (Hooijman et al, 2011). It has been proposed that (a) regulation of the transition between these states underlies the Frank-Starling law of the heart, and (b) dysregulation of the transition (destabilization of SRX) can lead to a form of heart failure known as hypertrophic cardiomyopathy (HCM). The structural basis of the SRX remains incompletely understood, but it has been proposed that it corresponds to the interacting heads motif (IHM) that has been observed by electron microscopy. To investigate directly the structural transition between DRX and SRX, we have developed a time-resolved FRET assay that measures the intermolecular distance between regulatory light chains on the two heads of myosin. The TRFRET data is best fit to two populations of Gaussian-distributed distances, suggesting that this FRET pair observes both the shorter distance predicted for the IHM (SRX) state and the longer distance predicted for the DRX state. This assay provides a structure-based assay for quantitating these states in functional muscle fibers, and it has the potential to create a high-throughput, high-precision assay for therapeutic development.