fMyBP-C is a vital regulator in young and aged fast skeletal muscle homeostasis [5-6 months old]

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. Overall design: 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 5-6 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（sMyBP-C）与快型（fMyBP-C），可与肌节内的肌球蛋白丝和肌动蛋白丝结合，调节收缩过程中的张力产生。二者在肌纤维中呈差异表达，快肌纤维中fMyBP-C的表达水平更高。然而，目前学界对病理状态下fMyBP-C表达的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功能意义。本研究使用22月龄老年雄性野生型及Mybpc2敲除（C2-/-）小鼠的骨骼肌样本，以明确fMyBP-C在衰老过程中的作用。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。通过免疫组织化学与电子显微镜技术，评估C2-/-敲除与衰老对肌纤维类型、直径、数量及整体肌肉结构的影响。通过体内与体外肌肉张力生成实验，检测C2-/-敲除与衰老对肌肉功能的影响。最后，通过RNA测序（RNA-seq）鉴定年轻与老年小鼠中C2-/-介导的肌肉功能障碍所涉及的失调分子通路。与同年龄野生型小鼠相比，老年雄性C2-/-小鼠的肌肉力量与耐力存在显著缺陷，并伴随肌纤维直径改变及肌肉稳态关键分子信号通路的异常。综上，fMyBP-C是年轻与老年快肌纤维中肌肉功能与稳态的重要调控因子。fMyBP-C的缺失会加剧衰老对肌肉结构与功能的损害。fMyBP-C有望成为干预衰老与疾病所致肌肉萎缩的治疗靶点。
实验整体设计：通过将Mybpc2基因第2至22号外显子用两侧带有两个LoxP位点的新霉素抗性基因盒（Neo cassette）进行靶向替换，构建Mybpc2敲除（C2-/-）小鼠模型。从5-6月龄雄性野生型与C2-/-小鼠（每组n=4）中分离胫骨前肌（Tibialis anterior，简称TA）。提取胫骨前肌中的RNA并进行RNA测序，以鉴定差异表达的分子特征。