Titin in Skeletal Muscle Health and Disease

人们对肌动蛋白在引起神经肌肉疾病中的重要性的认识迅速增加。 (钛病)，我们在这项工作中重点讨论的两种疾病是中心核肌病(CNM)和遗传性肌病 伴有早期呼吸衰竭(HMERF)。钛病通常表现为早发性肌肉无力和 呼吸困难，但对其潜在机制的理解受到我们仍然有限的 了解肌动蛋白在骨骼肌中的功能作用。肌动蛋白由肌肉的第三肌丝组成 并沿肌节横跨，从Z盘到M带。Titin的I带区域起到了分子弹簧的作用 这就产生了被动刚度，最近的研究表明，被动刚度会影响亚动力 最大激活级别。然而，目前还不知道肌动蛋白的僵硬对整个骨骼肌有多重要 健康，如果僵硬改变，可能会导致肌病。重要性可能很高，这是由我们的 先导性研究显示，患有CNM的牙病患者存在疯狂的Titin僵硬和主动张力降低。 目标1和目标2解决了改变基于Titin的僵硬如何影响被动和主动肌肉特性以及如果 它可能会导致疾病。我们将对牙病患者的活检组织进行机械研究(重点是 CNM)，并研究了两种不同的小鼠模型，在这两种模型中，Titin的弹簧区的僵硬程度是 增加(PEVK截断)或减少(RBM20失活)。工作假说是改变Titin的 弹力区会改变被动肌肉僵硬和主动张力，从而导致肌病。目标2 我还研究了一种新的小鼠模型，它模仿CNM，在Titin的弹簧区发生剪接位点突变，我们 将在RNA、蛋白质、结构和功能水平上研究功能后果。我们还学习 Titin的A波段段，特别是C区。这个区域还没有被研究过，它在临床上很重要 这就是发现大量致病突变的地方。我们在目标3中调查了一个突变 C区外显子343导致HMERF，这是一种伴有呼吸肌受累的肌病，可 致命的。利用我们制作的新型HMERF小鼠，对其发病机制进行了研究。 通过切除突变的Titin外显子343，外显子跳跃治疗HMERF的潜力 将会受到考验。凭借其基础科学和翻译目标以及深入和综合的方法， 这项申请旨在延续我们在尖端TiTIN研究方面的记录。强大的技术和 新的老鼠模型已经到位，试点数据支持指导性假设，我们的研究团队是 经验丰富。该提案将极大地提高对肌动蛋白生物学、肌动蛋白在肌肉疾病中的作用， 以及Titin作为治疗靶点的潜力。

There is a rapidly increasing awareness of the importance of titin in causing neuromuscular disorders (titinopathies), two of which we focus on in this work, centronuclear myopathy (CNM) and hereditary myopathy with early respiratory failure (HMERF). Titinopathies frequently present early-onset muscle weakness and respiratory difficulty but understanding their underlying mechanisms is held back by our still limited understanding of the functional roles of titin in skeletal muscle. Titin comprises the third myofilament of muscle and spans along the sarcomere, from Z-disk to M-band. Titin’s I-band region functions as a molecular spring that generates passive stiffness with recent studies indicating that passive stiffness affects active force at sub- maximal activation levels. However, it is not known how important titin’s stiffness is to overall skeletal muscle health and if altered stiffness can cause a myopathy. That the importance might be high is suggested by our pilot studies that reveal deranged titin stiffness and reduced active tensions in titinopathy patients with CNM. Aims 1 and 2 address how altering titin-based stiffness affects both passive and active muscle properties and if it can be disease-causing. We will perform mechanical studies on biopsies from titinopathy patients (focus on CNM) as well as study two contrasting mouse models in which the stiffness of titin’s spring region is either increased (PEVK truncation) or reduced (inactivation of Rbm20). The working hypothesis is that altering titin’s spring region alters both passive muscle stiffness and active tension and that this causes myopathy. Aim 2 also studies a novel mouse model that mimics CNM with splice site mutations in titin’s spring region and we will examine the functional consequences at the RNA, protein, structural and functional levels. We also study the A-band segment of titin, specifically the C-zone. This zone has not been studied, it is clinically important as this is where a large number of disease-causing mutations are found. We investigate in Aim 3 a mutation in the C-zone exon 343 which causes HMERF, a myopathy with respiratory muscle involvement that can be fatal. Using a novel HMERF mouse that we made the mechanistic basis of the disease will be studied. Through excision of the mutated titin exon 343, the therapeutic potential of exon skipping for treating HMERF will be tested. With its basic science and translational goals and its in-depth and integrative approach, this application seeks to continue our track record of cutting-edge titin research. Powerful techniques and novel mouse models are in place, pilot data support the guiding hypotheses, and our research team is highly experienced. The proposal will greatly enhance insights in titin biology, titin’s role in muscle disease, and titin’s potential as a therapeutic target.