Calcium binding to troponin C is required for activation of the myosin-containing thick filaments in rat cardiac trabeculae.

Contraction of the muscular walls of the heart is driven by an interaction between myosin motors from the thick filaments and actin sites in the thin filaments. Each heartbeat is triggered by calcium binding to troponin in the thin filaments, which unblocks the myosin-binding sites on actin. The strength and speed of contraction is also modulated by the availability of myosin motors, which are sequestered in a helical array on the surface of the thick filaments between heartbeats. The signalling pathway controlling release of the motors from this array during the heartbeat is unknown, but there are three general hypotheses: thick-filament mechano-sensing, thin-to-thick filament signalling, and direct calcium signalling to the thick filament. Here we tested the third hypothesis by replacing the native calcium-binding subunit of troponin (TnC) with a variant which cannot bind calcium. Demembranated trabeculae from rat heart containing this variant generated no active force on addition of calcium. We measured calcium-induced release of myosin motors from the sequestered state by X-ray diffraction and from the orientation of fluorescent probes on the myosin regulatory light chain. Both methods showed the expected calcium-dependent changes in the conformation of the myosin motors in trabeculae containing native TnC, but all these changes were abolished in those containing the TnC variant that cannot bind calcium. We conclude that thick filament activation in rat heart trabeculae is not due to direct binding of calcium to thick filaments, but is mediated by calcium activation of the thin filaments by mechano-sensing or thin-to-thick filament signalling.