Structural and Biochemical Kinetics of Cardiac Myosin and its Perturbation by a Known Heart Failure Drug Investigated with Transient Time-Resolved FRET

Abstract

Myosin cycles between pre- and post-powerstroke structural states during active force generation in muscle. We recently used transient time-resolved FRET to measure the kinetics and thermodynamics of this cycling, finding that in fast skeletal muscle myosin the actin-activated powerstroke occurs before actin-activated phosphate release. Here we investigate the structural kinetics of cardiac myosin during active ATPase cycling to determine (1) how the powerstroke and phosphate release steps are coordinated in a slower muscle myosin, and (2) how the heart-failure drug omecamtiv mecarbil (OM), that binds to the pocket near the force transducing beta-sheet of cardiac myosin, affects the structural kinetics of the cardiac myosin powerstroke. OM stabilizes the post-hydrolysis ADP.Pi bound biochemical state and should therefore (a) stabilize the pre-powerstroke structural state. Furthermore, it accelerates actin-activated phosphate release, so we reasoned it should (b) accelerate the powerstroke as well. Our results support hypothesis (a) but not (b): we find that OM increases the mole fraction of pre-powerstroke structural state in the absence of actin, but surprisingly, we find that OM causes the powerstroke to occur more slowly, after actin-induced phosphate release, rather than before as in the absence of the drug. These results suggest that OM changes the fundamental structural mechanics of the myosin powerstroke. Supported by NIH AR032961 & AR057220 (DDT), the Paul and Sheila Wellstone Muscular Dystrophy Center (JMM), American Heart Association Scientist Development Grant (JMM), and Graduate Excellence Fellowship-University of MN (JAR).