Abstract
We developed time-resolved fluorescence resonance energy transfer (FRET) sensors to detect the structural state of myosin in cardiac thick filaments. Cardiac myosin thick filaments are hypothesized to isomerize between an active state, termed RX (relaxed) that interacts with actin and generates force in the sarcomere, and an inactive state, termed SRX (super-relaxed) that does not bind actin and exhibits inhibited ATP hydrolysis compared to soluble myosin without actin. Regulation of the transition between these states is hypothesized to underlie the Frank-Starling law of the heart, and dysregulation of the transition to lead to genetically-driven hypertrophic cardiomyopathy; however, exactly how remains unclear, because the structural basis of the SRX is poorly understood. To investigate the structural transition between RX and SRX, we developed a time-resolved FRET assay that measures the intermolecular distance between two adjacent myosin regulatory light chains. Based on proposed structural models, formation of the SRX increases FRET while disruption decreases FRET. We tested this model with known modulators of the SRX biochemical kinetics, including temperature and ionic strength, and with two physiologically relevant SRX modulators, RLC phosphorylation and myosin-binding protein-C. This work was supported by National Institute of Health grants AR032961 and AR057220 (DDT), and American Heart Association Scientist Development Grant (JMM).
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