Slow myosin binding protein-C in skeletal muscle physiology

项目摘要：远端关节紊乱(DA)是一组不同类型的疾病，其特征是 与肌肉无力相关的先天性非进行性关节痉挛。取决于基因 涉及的和特定的突变，遗传是典型的常染色体显性与可变表达和 不完全的外露。目前的临床分类确定了11种不同的离散综合征，其中有几种 与肌节基因突变有关，包括慢骨骼肌球蛋白结合蛋白-C(MYBPC1)。 最近，MYBPC1中的一种纯合子隐性突变与一种严重的DA有关，即致命的先天性 肌挛缩症4型(LCCS4)。尽管DA综合征与特定基因的联系日益密切 突变，是骨骼肌无力的分子机制，可能导致残疾 人们对肌挛缩症知之甚少。由于这些机制是未知的，具体地说，人们对此知之甚少 SMyBP-C如何在体内调节肌肉功能，目前的治疗方法大多无效，并被归类为 对症理疗。 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 蛋白质C(MyBP-C)与健康和疾病。已知这些肌节特异的蛋白质可以调节 通过调节肌动球蛋白功能的横纹肌收缩能力。存在三个MyBP-C类似物，即慢 骨骼MyBP-C(sMyBP-C)、快速骨骼MyBP-C(fMyBP-C)和心肌MyBP-C，由不同的基因编码。 这项提议的具体目标是定义强调sMyBP突变的生理机制- C会导致肌肉功能障碍和痉挛。在我们的初步研究中，我们确定了那些 都是纯合的全局sMyBP-C空(Mybpc1-/-)，类似于人类LCCS4的表型，都在 出生第一天，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(Mybpc1fl/fl)导致 分别在出生后和成年阶段出现明显的肌肉无力。最后，我们在转基因小鼠身上展示了 人α-骨架肌动蛋白启动子sMyBP-C控制下的Mybpc1Tg高表达 FMyBP-C损害快速肌型功能。 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 在慢肌和快肌两种类型下都能发挥作用。计划中的实验将系统地定义(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 对于出生后和成年阶段的骨骼肌功能，以及(Iii)sMyBP-C和fMyBP-C反式补充 彼此之间。我们预计，解决这些关键问题将推动对如何 SMyBP-C在不同发育阶段调节骨骼肌生理。因此，这项提议将 确定治疗目标以改善患有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.