核膜蛋白nesprin基因突变导致扩张型心肌病的分子机制研究

Mutations in the LMNA gene, encoding an important nuclear envelope (NE) protein lamin A/C, is the most common genetic cause of dilated cardiomyopathy (DCM). Recent studies revealed that LMNA mutations cause the aberrant activation of mitogen-activated protein kinases (MAPKs), contributing to the pathogenesis of DCM. However, the underlying molecular mechanism remains unclear. We have recently identified three novel nesprin-1 mutations in DCM patients in our last project funded by NSFC. Nesprins are a family of multi-isomeric scaffolding proteins that were originally identified at the NE, where they bind to lamin A/C, emerin and SUN-domain containing proteins, to form the LInker of Nucleoskeleton-and-Cytoskeleton (LINC) complex that connects the NE to the actin cytoskeleton, which plays a curcial role in the mechanotransduction signaling pathway. Therefore, we hypothesis that mutations in either lamin A/C or nesprin genes may impair mechanotransduction via altering the LINC complex, which leads to aberrant activation of MAPKs and ultimately DCM. In this project, we prepare to investigate if these novel nesprin-1 mutants will cause similar defects on the nuclear structure, leading to disruption of the LINC complex and aberrant activation of MAPKs, which may reveal underlying mechanisms that contribute to the development of muscle cell dysfunction and DCM. This will provide information that can be used to design new tools for early genetic diagnosis or prognosis as well as novel therapies.

研究发现LMNA基因突变导致的核膜蛋白lamin A/C缺陷是扩张型心肌病（DCM）最常见的遗传学病因。本课题组在已结题自然科学基金课题中发现核膜蛋白nesprin的突变也与DCM相关。本课题中我们将深入探讨nesprin缺陷是否通过损害机械传导信号通路引起丝裂原激活蛋白激酶系统（MAPKs）信号系统异常激活，并最终导致DCM。本研究将建立nesprin突变的体外和体内模型，探索突变是否影响nesprin与其结合蛋白的亲和力，是否影响核膜的结构稳定性，是否通过MAPKs信号系统异常激活导致DCM表型。在此基础上通过牵拉实验进一步探明机械传导通路是否是核膜蛋白缺陷引起MAPKs激活的共通通路。本课题的结果将有助于深入阐明核膜蛋白缺陷在DCM发病机制中的作用，为DCM的早期诊断、预后判断和新药物靶点研究提供理论依据。

课题组前期突变筛查研究中发现核膜蛋白nesprin-1基因的突变可能导致DCM，但具体致病机制尚不清楚。在本项目研究中，我们通过质粒转染和病毒感染分别在细胞系和原代心肌细胞中构建了nesprin-1蛋白缺陷的细胞模型。通过免疫荧光染色发现nesprin-1缺陷导致核膜结构的破坏。通过GST pull down等生化试验发现突变降低了nesprin-1和核膜蛋白lamin A/C、SUN-2的结合力，这可能是突变破坏核膜结构的分子机制。进一步的研究显示nesprin-1突变通过干扰核膜结构和功能导致ERK信号通路的异常激活。此外，本项目首次发现突变干扰nesprin-1与核外膜的KLC-1/2结合，也导致肌原细胞分化成熟的能力减低。本研究的结果初步阐明了核膜蛋白nesprin-1突变导致DCM的机制，为进一步DCM新疗法的研发提供了重要的理论基础。