Kalakoutis Michaeljohn, Wang Yanhong, Smith Emma, Arcidiacono Alice, Hill Cameron, Juma Samina, Fukutani Atsuki, Brunello Elisabetta, Fusi Luca, Irving Malcolm
Randall Centre for Cell and Molecular Biophysics and British Heart Foundation Centre of Research Excellence, King's College London, London, UK.
Randall Centre for Cell and Molecular Biophysics and British Heart Foundation Centre of Research Excellence, King's College London, London, UK.
J Mol Cell Cardiol. 2025 Jul 1;205:129-138. doi: 10.1016/j.yjmcc.2025.06.012.
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.
心脏肌壁的收缩是由粗肌丝上的肌球蛋白马达与细肌丝上的肌动蛋白位点之间的相互作用驱动的。每次心跳都是由钙离子与细肌丝中的肌钙蛋白结合触发的，这会解除肌动蛋白上肌球蛋白结合位点的阻碍。收缩的强度和速度也受到肌球蛋白马达可用性的调节，在心跳间期，这些马达以螺旋阵列的形式隔离在粗肌丝表面。控制心跳期间这些马达从该阵列释放的信号通路尚不清楚，但有三个普遍的假设：粗肌丝机械传感、细肌丝到粗肌丝信号传导以及直接向粗肌丝的钙信号传导。在这里，我们通过用一种不能结合钙的变体取代肌钙蛋白（TnC）的天然钙结合亚基来测试第三个假设。含有这种变体的大鼠心脏脱膜小梁在添加钙时不会产生主动力。我们通过X射线衍射以及肌球蛋白调节轻链上荧光探针的取向来测量钙诱导的肌球蛋白马达从隔离状态的释放。两种方法都显示，在含有天然TnC的小梁中，肌球蛋白马达构象出现了预期的钙依赖性变化，但在含有不能结合钙的TnC变体的小梁中，所有这些变化都被消除了。我们得出结论，大鼠心脏小梁中的粗肌丝激活不是由于钙直接与粗肌丝结合，而是由机械传感或细肌丝到粗肌丝信号传导介导的细肌丝钙激活引起的。
