Calcium- and myosin-dependent changes in troponin structure during activation of heart muscle

Abstract

Each heartbeat is triggered by a pulse of intracellular calcium ions which bind to troponin on the actin-containing thin filaments of heart muscle cells, initiating a change in filament structure that allows myosin to bind and generate force. We investigated the molecular mechanism of calcium regulation in demembranated trabeculae from rat ventricle using polarised fluorescence from probes on troponin C (TnC). Native TnC was replaced by double-cysteine mutants of human cardiac TnC with bifunctional rhodamine attached along either the C helix, adjacent to the regulatory Ca2+-binding site, or the E helix in the IT arm of the troponin complex. Changes in the orientation of both troponin helices had the same steep Ca2+-dependence as active force production, with a Hill coefficient (nH) close to 3, consistent with a single co-operative transition controlled by Ca2+ binding. Complete inhibition of active force by 25 μM blebbistatin had very little effect on the Ca2+-dependent structural changes and in particular did not significantly reduce the value of nH. Binding of rigor myosin heads to thin filaments following MgATP depletion in the absence of Ca2+ also changed the orientation of the C and E helices, and addition of Ca2+ in rigor produced further changes characterized by increased Ca2+ affinity but nH close to 1. These results show that, although myosin binding can switch on thin filaments in rigor conditions, it does not contribute significantly under physiological conditions. The physiological mechanism of co-operative Ca2+-regulation of cardiac contractility must therefore be intrinsic to the thin filaments.