Structural Changes in the Myosin Motors Induced by Stretch and Phosphorylation of the Myosin Regulatory Light Chain in Rat Cardiac Trabeculae

Abstract

The contractility of cardiac muscle is potentiated by phosphorylation of the myosin regulatory light chain (RLC) and by length-dependent activation, but the molecular pathways mediating these effects remain unclear. Here we studied the role of structural changes in the thick filament in the activation of cardiac muscle by stretch and RLC phosphorylation. We used fluorescence polarization from bifunctional sulphorhodamine probes on the N- and C-lobes of the myosin RLC to monitor changes in the orientation of the myosin motors induced by increasing the sarcomere length in the range 2.0-2.3 µm in relaxed and partially calcium-activated demembranated cardiac trabeculae, at different levels of RLC phosphorylation. We show that under relaxing or diastolic conditions at near physiological temperature and lattice spacing the RLC lobe orientations are consistent with about one third of the myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments. This folded conformation is disrupted by cooling relaxed trabecula, but not by RLC phosphorylation or stretch at diastolic [Ca2+]. These results indicate that in near-physiological conditions the myosin filament is not switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in diastole, as suggested previously. However, stretching cardiac trabecula at near-systolic [Ca2+] triggers a stress-dependent activation of the thick filament and a delayed increase in active force. Physiological levels of RLC phosphorylation potentiate both the stress-dependent release of myosin motors from the folded state and the stretch-activated force. We conclude that the increase in heart contractility induced by RLC phosphorylation and stretch can be explained by an inter-filament signaling pathway involving both thin filament sensitization and thick filament mechano-sensing (Supported by Wellcome Trust, BHF, UK).