fMyBP-C is a vital regulator in young and aged fast skeletal muscle homeostasis

Skeletal Myosin binding protein-C (MyBP-C) paralogs, slow (sMyBP-C) and fast (fMyBP-C) interacts with myosin and actin filaments within sarcomeres, modulating the force development during contraction. These display differential expression in muscle fibres, with fMyBP-C higher in fast twitch fibres. However, our knowledge about the changes in the fMyBP-C expression in diseased conditions and its role in skeletal muscle aging is lacking. In this study we use mice model lacking fMyBP-C to understand its significance in skeletal muscle physiology. Skeletal muscle samples from wild type and Mybpc2 knockout (C2-/-)old male mice (22 months) were used to define the role of fMyBP-C in aging. Western immunoblotting was employed to analyze the expression of fMyBP-C and sMyBP-C, and phosphorylation status of sMyBP-C. The impact of C2-/- and aging on the fiber type, size, and number as well as general muscle structure was assessed by immunohistochemistry and electron microscopy. The functional effect of C2-/- and aging was measured in terms of in vivo and ex vivo muscle force generation. Lastly, RNA sequencing was performed to identify the molecular pathways dysregulated in the C2-/- mediated muscle dysfunction in young and old mice. The aged male C2-/- mice compared to their wildtype counterparts, display significant deficits in muscle strength and endurance, accompanied by changes in muscle fiber size and molecular signaling pathways critical for muscle homeostasis.Thus, fMyBP-C is an important regulator of muscle function and homeostasis in the young and aged fast-twitch muscle fibers. The absence of fMyBP-C aggravates the effect of aging on muscle structure and function. fMyBP-C has the potential to be a therapeutic target to modulate muscle wasting caused by aging and disease. Mybpc2 KO mouse model (C2-/-) was generated by the targeted replacement of Mybpc2 exon 2 to 22 with a Neo cassette flanked by two LoxP sites. The Tibialis anterior muscle dissected from 22 months old male Wildtype and C2-/- (n=4 in each group). RNA extracted from TA muscles are subjected to RNA Sequencing to determine the altered molecular signatures.骨骼肌肌球蛋白结合蛋白-C（MyBP-C）的旁系同源物——慢型肌球蛋白结合蛋白-C（sMyBP-C）与快型肌球蛋白结合蛋白-C（fMyBP-C），可与肌节（sarcomeres）内的肌球蛋白丝和肌动蛋白丝结合，调节肌肉收缩过程中的力产生。二者在肌纤维中存在差异表达：快肌纤维（fast twitch fibres）中fMyBP-C的表达水平更高。然而，目前学界对疾病状态下fMyBP-C的表达变化及其在骨骼肌衰老中的作用仍知之甚少。本研究采用fMyBP-C敲除小鼠模型，探究其在骨骼肌生理学中的核心作用。实验选取22月龄的雄性野生型与Mybpc2基因敲除（C2-/-）老年小鼠的骨骼肌样本，旨在明确fMyBP-C在衰老进程中的功能。采用蛋白质免疫印迹（Western immunoblotting）技术，分析fMyBP-C与sMyBP-C的表达水平，以及sMyBP-C的磷酸化状态；通过免疫组织化学（immunohistochemistry）与电子显微镜（electron microscopy）技术，评估C2-/-突变与衰老对肌纤维类型、尺寸、数量及整体肌肉结构的影响；通过体内（in vivo）与体外（ex vivo）肌肉力产生实验，测定C2-/-突变与衰老对肌肉功能的影响。最后，通过RNA测序（RNA sequencing），鉴定年轻与老年小鼠中C2-/-介导的肌肉功能障碍相关的失调分子通路。与同年龄段野生型小鼠相比，老年雄性C2-/-小鼠的肌肉力量与耐力存在显著缺陷，同时伴随肌纤维尺寸改变以及调控肌肉稳态的关键分子信号通路异常。综上，fMyBP-C是年轻与老年快肌纤维中肌肉功能及稳态的重要调节因子；fMyBP-C的缺失会加剧衰老对肌肉结构与功能的损害，其有望成为干预衰老与疾病所致肌肉萎缩的治疗靶点。本研究中的Mybpc2基因敲除（C2-/-）小鼠模型，通过将Mybpc2基因外显子2至22替换为两侧带有两个LoxP位点的Neo基因盒构建得到。实验采集了22月龄雄性野生型与C2-/-小鼠的胫骨前肌（Tibialis anterior，TA肌，每组n=4），从胫骨前肌中提取的RNA将进行RNA测序，以确定其中的差异分子特征。