Calcium- and myosin-dependent changes in troponin structure during activation of heart muscle.
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 polarized 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 Ca(2+)-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 Ca(2+) dependence as active force production, with a Hill coefficient (n(H)) close to 3, consistent with a single co-operative transition controlled by Ca(2+) binding. Complete inhibition of active force by 25 microM blebbistatin had very little effect on the Ca(2+)-dependent structural changes and in particular did not significantly reduce the value of n(H). Binding of rigor myosin heads to thin filaments following MgATP depletion in the absence of Ca(2+) also changed the orientation of the C and E helices, and addition of Ca(2+) in rigor produced further changes characterized by increased Ca(2+) affinity but with n(H) 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 Ca(2+) regulation of cardiac contractility must therefore be intrinsic to the thin filaments.
Sun
Yin-Biao
YB
Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, UK. yin-biao.sun@kcl.ac.uk
Lou
Fang
F
Irving
Malcolm
M
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009200:Myocardial Contraction / Q000502:physiology; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D017433:Protein Structure, Secondary; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D019209:Troponin C; D009218:Myosins; D002118:Calcium