Detection of Ultrafast Mechanical Transitions in β-Cardiac Myosin using High-Speed Optical Trapping

Abstract

The β isoform of myosin II (MYH7) is the primary myosin heavy chain present in both slow skeletal muscle and the cardiac ventricles of large mammals. In cardiac and skeletal muscle, strong binding of myosin to the thin filament plays an important role in the cooperative activation of contraction. Studying the weak-to-strong transition and the initiation of the force-producing power stroke has been difficult using single molecule techniques because standard optical trapping methods require 10-15 ms to detect actin-myosin interactions. This latency is much longer than the expected time from initial actin-myosin interaction to strong binding and force production. In this work we study full-length, tissue-purified, porcine β-cardiac myosin using an ultrafast optical trapping technique that allows the detection of actin-myosin binding events within 1 ms. This improved time resolution reveals three populations of acto-myosin attachments with lifetimes that differ by more than 10-fold, likely representing three different biochemical and/or mechanical states. The slowest of these rates is ATP dependent at low MgATP concentrations and corresponds well with the expected rate of ATP-induced dissociation. The populations with shorter lifetimes likely represent dissociation from actomyosin states that precede ATP-induced dissociation. The force dependence of these lifetimes and the relative amplitude of each phase reveal information about mechano-chemical transitions in cardiac myosin that have not previously been probed in the single molecule regime. In addition, ensemble averages of individual attachment events reveal the amplitude and timing of both sub-steps of β-cardiac myosin's power stroke. Through these ensemble averages, we can separately resolve the pre-powerstroke/actin-bound state and the post-powerstroke state, representing the first single-molecule observation of the initial actin displacement by cardiac myosin under load. Supported by NIH grant P015GM087253.