Stretch-Induced Activation of the Myosin Motors on the Thick Filament in Rat Cardiac Trabeculae

Abstract

The contractility of cardiac muscle is regulated by thick-filament based mechanisms in addition to the classical calcium/thin-filament mediated mechanisms. Here we studied the role of structural changes of the thick filament in length-dependent activation in cardiac muscle. We used fluorescence polarization from bifunctional sulphorhodamine probes on the N- and C-lobes of the myosin regulatory light chain (RLC) to monitor changes in the orientation of the myosin motors induced by increasing sarcomere length in relaxed and partially calcium-activated demembranated trabeculae. Under relaxing conditions at near physiological temperature and lattice spacing the myosin motors are roughly parallel to the filament axis, consistent with the OFF structure of the thick filament. Cooling of the relaxed trabecula induced a more perpendicular orientation of the myosin motors indicating disruption of the OFF-state. Application of a staircase stretch protocol in the sarcomere length range 2.0-2.3 μm (25°C, 3% dextran T500) at pCa 9.0 also induced a more perpendicular orientation of the myosin motors, but despite an increase in passive tension to ∼30% of the maximum active force (T0), the orientation change was only ∼4% of that associated with full calcium activation. Larger stretch-induced orientation changes were observed at pCa 7 (∼7%) and at pCa 6.6 (∼20%), the latter accompanied by an increase in active force of ∼0.05 T0. RLC phosphorylation further increased the stretch-induced orientation changes to ∼14% and ∼33% respectively, and the active force response at pCa 6.6 increased to ∼0.2 T0. These results indicate that increasing sarcomere length in the physiological range at diastolic calcium concentration induces activation of the myosin motors and that this effect is enhanced by RLC phosphorylation, suggesting that changes in thick filament structure may mediate length-dependent activation. Supported by Wellcome Trust and BHF, UK.