Fast skeletal myosin binding protein-C regulates fast skeletal muscle contraction

Fast skeletal myosin binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, is associated with distal arthrogryposis, while fMyBP-C protein is reduced in diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2-/-) and characterized it, both in vivo and in vitro. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar-flexor muscle strength were significantly decreased in C2-/- compared to WT. Peak isometric tetanic force (Po) and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2-/- mice. In EDL muscles of C2-/- mice, small-angle X-ray diffraction revealed significant increase in equatorial intensity ratio (I1.1/I1.0) during contraction, indicating a greater degree of myosin head shift towards actin while MLL4 layer-line intensity was decreased at rest, indicating less ordered myosin heads at rest. Interfilament lattice spacing was also significantly increased in C2-/- EDL muscle compared to WT. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca2+ activations, myofilament calcium sensitivity, sinusoidal stiffness in skinned EDL muscle fibers from C2-/- mice. Finally, C2-/- muscles displayed disruption of inflammatory and regenerative genes and increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast twitch muscle. Comparision of RNA sequence between wild type (WT ) and C2-/- EDL fiber

快骨骼肌肌球蛋白结合蛋白C（fast skeletal myosin-binding protein C，fMyBP-C）是三类肌球蛋白结合蛋白C旁系同源物之一，主要在快骨骼肌中表达。编码fMyBP-C的基因MYBPC2发生突变与远端关节弯曲症相关，而病变肌肉中fMyBP-C蛋白水平显著降低。然而，fMyBP-C在骨骼肌中的功能与结构作用仍未明确。为填补该研究空白，我们构建了纯合fMyBP-C敲除小鼠（C2⁻/⁻），并分别在体内与体外对其进行了表型表征。结果显示，敲除fMyBP-C对肌肉重量、肌纤维类型、横截面积及肌节超微结构均无显著负面影响。但与野生型（wild type，WT）小鼠相比，C2⁻/⁻小鼠的抓力与跖屈肌力量均显著下降。从C2⁻/⁻小鼠体内分离的趾长伸肌（extensor digitorum longus，EDL）的等长强直收缩峰值力（Po）与等张收缩速度均显著降低。对C2⁻/⁻小鼠EDL肌开展小角X射线衍射分析发现，收缩过程中赤道衍射强度比（I1.1/I1.0）显著升高，提示肌球蛋白头部向肌动蛋白方向的移位程度更大；而静息状态下MLL4层线衍射强度降低，提示静息状态下肌球蛋白头部的排列有序性下降。此外，与WT小鼠相比，C2⁻/⁻小鼠EDL肌的肌丝间晶格间距亦显著增大。与上述研究结果一致，我们在透化处理的C2⁻/⁻小鼠EDL肌纤维中观察到：钙离子激活过程中的稳态等长收缩力、肌丝钙敏感性以及正弦刚度均显著降低。最后，C2⁻/⁻肌肉表现出炎症与再生相关基因的表达紊乱，且在机械超负荷条件下肌肉损伤程度加剧。综上，本研究结果表明，fMyBP-C对于快肌纤维的最大收缩速度与收缩力、肌节完整性以及钙敏感性均至关重要。野生型（WT）与C2⁻/⁻ EDL肌纤维的RNA序列对比