Slow myosin binding protein-C in skeletal muscle physiology

项目总结：远端关节弯曲（DA）是一组异质性疾病， 先天性非进行性关节挛缩伴肌肉无力取决于基因 涉及和特定突变，遗传通常是常染色体显性的，具有可变表达， 不完全昏迷目前的临床分类确定了11种不同的离散综合征， 与肌节基因突变相关，包括慢骨骼肌球蛋白结合蛋白-C（MYBPC 1）。 最近，MYBPC 1的纯合隐性突变与一种严重的DA有关， 挛缩综合征4型（LCCS 4）。尽管DA综合征与特定遗传因素的相关性越来越大， 突变，骨骼肌无力的分子机制，可能导致残疾 对挛缩的了解很少。由于这些机制是未知的，具体来说， sMyBP-C如何在体内调节肌肉功能，目前的疗法在很大程度上是无效的， 对症物理治疗 我们研究计划的总体长期目标是确定肌球蛋白结合的贡献， 蛋白C（MyBP-C）蛋白在健康和疾病中的作用。已知这些肌节特异性蛋白调节 通过调节肌动球蛋白功能来增强横纹肌收缩力。存在三种MyBP-C旁系同源物，即慢 骨骼肌MyBP-C（sMyBP-C）、快速骨骼肌MyBP-C（fMyBP-C）和心脏MyBP-C，并由不同的基因编码。 该提案的具体目标是定义强调sMyBP突变如何发生的生理机制， C导致肌肉功能障碍和挛缩。在我们的初步研究中，我们确定， 是纯合的全局sMyBP-C null（Mybpc 1-/-），类似于人LCCS 4表型，均在 出生第一天，出现继发于肌肉萎缩的震颤。我们证明了肌肉肌酸 激酶Cre-和人α-骨骼肌动蛋白-Cre/他莫昔芬介导的sMyBP-C消融（Mybpc 1fl/fl）导致 在出生后和成年阶段分别出现明显的肌肉无力。最后，我们在转基因小鼠中发现， 在sMyBP-C取代的人α-骨架肌动蛋白启动子的控制下过表达Mybpc 1 Tg fMyBP-C损害快肌型功能。 基于这些数据，我们假设sMyBP-C作为横纹肌形成的关键调节因子， 在慢肌和快肌类型中都起作用。计划中的实验将系统地确定（i） sMyBP-C是产前和围产期肌肉正常形成所必需的，（ii）sMyBP-C是必需的 在出生后和成年阶段的骨骼肌功能，和（iii）sMyBP-C和fMyBP-C反式补体 对方.我们预计，解决这些关键问题将推动机械理解如何 sMyBP-C在发育阶段调节骨骼肌生理学。因此，本提案将 确定治疗靶点以改善患有DA疾病的患者的肌肉功能。

PROJECT SUMMARY: The distal arthrogryposes (DA) are a heterogeneous group of disorders characterized by congenital nonprogressive joint contractures associated with muscle weakness. Depending on the gene involved and the specific mutation, inheritance is typically autosomal dominant with variable expression and incomplete penetrance. Current clinical classification identifies eleven different discrete syndromes with several associated with mutations in sarcomere genes including slow skeletal myosin binding protein-C (MYBPC1). Recently, a homozygous recessive mutation in MYBPC1 was linked to a severe form of DA, lethal congenital contracture syndrome type 4 (LCCS4). Despite the increasing association of DA syndromes with specific genetic mutations, molecular mechanisms that underlie skeletal muscle weakness that presumably lead to disabling contractures are poorly understood. As these mechanisms are unknown and, specifically, little is known about how sMyBP-C regulates muscle function in vivo, current therapies are largely ineffective and relegated to symptomatic physical therapy. The overall long-term goal of our research program has been to define the contribution of the myosin binding protein-C (MyBP-C) proteins in health and disease. These sarcomeric-specific proteins are known to regulate striated muscle contractility via modulating actomyosin function. Three MyBP-C paralogs exist, namely slow skeletal MyBP-C (sMyBP-C), fast skeletal (fMyBP-C), and cardiac MyBP-C, and encoded by separate genes. The specific goal of this proposal is to define the physiologic mechanisms underlining how mutations in sMyBP- C lead to muscle dysfunction and contractures. In our preliminary studies, we determined that mouse pups that are homozygous global sMyBP-C null (Mybpc1-/-), similar to the human LCCS4 phenotype, all died within the first day of birth and exhibited tremors secondary to muscle atrophy. We demonstrated that muscle creatine kinase Cre- and human a-skeletal actin-Cre/Tamoxifen-mediated sMyBP-C ablation (Mybpc1fl/fl) resulted in significant muscle weakness in postnatal and adult stages, respectively. Finally, we showed in transgenic mice overexpressing Mybpc1Tg under the control of the human a-skeletal actin promoter that sMyBP-C replaces fMyBP-C impairing fast muscle type function. Based on these data, we hypothesize that sMyBP-C acts as a key regulator of striated muscle formation and function in both slow and fast muscle types. The planned experiments will systematically define whether (i) sMyBP-C is essential for normal formation of muscle in prenatal and perinatal stages, (ii) sMyBP-C is required for skeletal muscle function in postnatal and adult stages, and (iii) sMyBP-C and fMyBP-C transcomplement each other. We anticipate that addressing these key questions will drive mechanistic understanding of how sMyBP-C regulates skeletal muscle physiology across developmental stages. Consequently, this proposal will identify therapeutic targets to improve muscle function in those afflicted with DA diseases.