Risi Cristina M, Landim-Vieira Maicon, Belknap Betty, Chase P Bryant, Pinto Jose R, Galkin Vitold E
Department of Biomedical and Translational Sciences, Macon & Joan Brock Virginia Health Sciences at Old Dominion University, Norfolk, VA 23507, USA.
Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL, USA.
J Mol Cell Cardiol. 2025 Jul;204:55-67. doi: 10.1016/j.yjmcc.2025.05.005. Epub 2025 May 22.
Cardiac muscle contraction/relaxation cycle depends on the rising and falling Ca levels in sarcomeres that control the extent of interactions between myosin-based thick and actin-based thin filaments. Cardiac thin filament (cTF) consists of actin, tropomyosin (Tm) that regulates myosin binding to actin, and troponin complex that governs Tm position upon Ca-binding. Troponin has three subunits - Ca-binding troponin C (TnC), Tm stabilizing troponin T (TnT), and inhibitory troponin I (TnI). TnT N-terminus (TnT1) interactions with actin stabilize the inhibited state of cTF. TnC, TnI, and Tm work in concert to control actomyosin interactions. Cryo-electron microscopy (cryo-EM) provided factual structures of healthy cTF, but structures of cTF carrying missense mutations linked to human cardiomyopathy are unknown. Variant Ile79Asn in human cardiac TnT (TnT-I79N) increases myofilament Ca sensitivity and slows cross-bridge kinetics, leading to severe hypertrophic/restrictive cardiomyopathy. Here, we used TnT-I79N mutation as a tool to examine the role of TnT1 in the complex mechanism of cTF regulation. Comparison of the cryo-EM structures of murine wild type and TnT-I79N native cTFs at systolic Ca levels (pCa = 5.8) demonstrates that TnT-I79N causes 1) dissociation of the TnT1 loop from its actin interface that results in Tm release to a more activated position, 2) reduced interaction of TnI C-terminus with actin-Tm, and 3) increased frequency of Ca-bound regulatory units. Our data indicate that the TnT1 loop is a crucial element of the allosteric regulatory network that couples Tn subunits and Tm to maintain adequate cTF response to physiological Ca levels during a heartbeat.
心肌收缩/舒张周期取决于肌节中钙离子水平的升降，钙离子水平控制着基于肌球蛋白的粗肌丝和基于肌动蛋白的细肌丝之间相互作用的程度。心脏细肌丝（cTF）由肌动蛋白、调节肌球蛋白与肌动蛋白结合的原肌球蛋白（Tm）以及在结合钙离子时控制Tm位置的肌钙蛋白复合体组成。肌钙蛋白有三个亚基——结合钙离子的肌钙蛋白C（TnC）、稳定Tm的肌钙蛋白T（TnT）和抑制性肌钙蛋白I（TnI）。TnT的N端（TnT1）与肌动蛋白的相互作用稳定了cTF的抑制状态。TnC、TnI和Tm协同作用以控制肌动球蛋白的相互作用。冷冻电子显微镜（cryo-EM）提供了健康cTF的真实结构，但携带与人类心肌病相关错义突变的cTF结构尚不清楚。人类心脏TnT中的Ile79Asn变体（TnT-I79N）增加了肌丝对钙离子的敏感性并减缓了横桥动力学，导致严重的肥厚性/限制性心肌病。在这里，我们使用TnT-I79N突变作为工具来研究TnT1在cTF调节复杂机制中的作用。对收缩期钙离子水平（pCa = 5.8）下小鼠野生型和TnT-I79N天然cTF的冷冻电子显微镜结构进行比较表明，TnT-I79N导致：1）TnT1环与其肌动蛋白界面解离，导致Tm释放到更活跃的位置；2）TnI C端与肌动蛋白-Tm的相互作用减少；3）结合钙离子的调节单位频率增加。我们的数据表明，TnT1环是变构调节网络的关键要素，该网络将Tn亚基和Tm耦合起来，以在心跳期间维持cTF对生理钙离子水平的适当反应。
