Novel MYBPC1 mutations cosegregate with a myopathy associated with muscle weakness, hypotonia and tremor

摘要 肌球蛋白结合蛋白-C（MyBP-C）包括与肌球蛋白结合蛋白直接相互作用的辅助蛋白家族。 粗肌球蛋白和细肌动蛋白丝。已经表征了三种不同的同种型，包括 心脏（c）、慢速（s）骨骼和快速（f）骨骼。在过去的四十年里，许多研究都集中在 cMyBP-C的生物学主要是由于其在心脏病中的高突变率。反倒是 骨骼亚型的调节和作用主要是由于它们的结构相似性而推断出来的， 与cMyBP-C共享。我们小组一直在研究sMyBP-C的生物学，旨在了解其调控， 角色和疾病关联。我们的发现非常新颖和有趣。首先，我们发现MYBPC1， 编码sMyBP-C的基因被大量剪接，产生多种变体，这些变体可以在细胞中共表达。 相同的肌肉和肌纤维第二，选择性剪接插入的存在影响了细胞的能力。 NH2和COOH末端结合肌动蛋白和肌球蛋白，并调节肌动球蛋白横桥的形成。 体外第三，sMyBP-C的NH 2-末端经历广泛的PKA-和PKC-介导的磷酸化， 在疾病中发生改变。第四，sMyBP-C具有结构和调节作用， 粗肌球蛋白丝的组织和维持在其调节交叉- 桥牌自行车第五，sMyBP-C的NH 2-末端M-基序中存在4个新的显性错义突变， 与一种新的肌病共分离，其特征在于肌肉无力、张力减退、面部和身体 畸形和高频不规则震颤分子建模和生物化学研究表明， 四种肌病突变不同地影响sMyBP-C的NH 2-末端结合肌球蛋白的能力， 以及M基序的结构和稳定性。因此，我们假设四个MYBPC1突变可能 差异性地改变sMyBP-C的生化和生物物理特性，损害其结构和 调节作用，但引起类似的肌病表型。我们进一步提出，突变的sMyBP-C导致 形成不正常的和放松管制的跨桥，这除了造成力量不足外， 产生潜在的肌肉无力，作为观察到的震颤的主要起搏器。我们的目标 建议全面研究这种新型MYBPC1相关肌病的发病机制 使用复杂的体外方法（目标1）和新的临床前模型（目标2和3）的组合。 所提出的研究在发现科学方面具有非常重要的意义，因为我们将机械地检查 这种肌病的病因，并在准确疾病的转化/临床研究方面具有影响力 诊断以及适当和有效的治疗设计。

Abstract Myosin Binding Protein-C (MyBP-C) comprises a family of accessory proteins that directly interact with both thick myosin and thin actin filaments. Three distinct isoforms have been characterized, including the cardiac (c), slow (s) skeletal and fast (f) skeletal. During the last forty years, numerous studies have focused on the biology of cMyBP-C primarily due to its high mutational prevalence in heart disease. On the contrary, the regulation and roles of the skeletal isoforms have been mainly inferred due to the structural similarity they share with cMyBP-C. Our group has been studying the biology of sMyBP-C aiming to understand its regulation, roles, and disease association. Our findings are highly novel and intriguing. First, we found that MYBPC1, the gene that encodes sMyBP-C, is heavily spliced giving rise to multiple variants that can be co-expressed in the same muscle and myofiber. Second, the presence of alternatively spliced insertions affects the ability of the NH2 and COOH termini to bind actin and myosin, and regulate the formation of actomyosin crossbridges in vitro. Third, the NH2-terminus of sMyBP-C undergoes extensive PKA- and PKC-mediated phosphorylation, which is altered in disease. Fourth, sMyBP-C has both structural and regulatory roles with its structural role in the organization and maintenance of thick myosin filaments preceding its regulatory role in modulating cross- bridge cycling. Fifth, four novel, dominant, missense mutations located in the NH2-terminal M-motif of sMyBP-C co-segregate with of a new myopathy characterized by muscle weakness, hypotonia, facial and body deformities, and high-frequency irregular tremor. Molecular modeling and biochemical studies indicated that the four myopathic mutations differentially affect the ability of the NH2-terminus of sMyBP-C to bind myosin, and the structure and stability of the M-motif. We therefore hypothesize that the four MYBPC1 mutations may differentially alter the biochemical and biophysical properties of sMyBP-C compromising its structural and regulatory roles, yet elicit similar myopathic phenotypes. We further propose that mutant sMyBP-C results in the formation of abnormal and deregulated cross-bridges, which in addition to causing a deficit in force production underlying muscle weakness, act as the primary pacemaker of the observed tremor. The goal of our proposal is to comprehensively study the pathogenesis of this novel form of MYBPC1-associated myopathy using a combination of sophisticated in vitro approaches (Aim 1) and novel preclinical models (Aims 2 & 3). The proposed studies are highly significant in terms of discovery science as we will mechanistically examine the etiologies of this myopathy, and impactful in terms of translational/clinical research for accurate disease diagnosis as well as appropriate and effective treatment design.