Dchs1 and the Septin Cytoskeleton: a Molecular and Developmental Etiology Underlying Mitral Valve Prolapse

摘要 二尖瓣脱垂(MVP)是最常见的心脏瓣膜疾病之一，影响约2-3%的心脏瓣膜疾病 人类人口。目前还没有有效的非手术治疗MVP的方法，治疗的努力一直很多。 由于对其根本原因的不完全理解而受到阻碍。然而，我们现在有令人信服的基因 和功能证据，极大地促进了我们对MVP发病机制的理解。我们的小组是 首先通过鉴定非典型钙粘附素基因DCHS1的突变来确定MVP的遗传原因， 在多个患有非综合征性MVP的家庭中，并已将疾病的起源追溯到胎儿瓣膜缺陷 形态发生。DCHS1缺乏的不同的功能和分子后果目前还没有 已知的，但最近的双杂交研究揭示了DCHS1，Lix-1样(LIX1L)， 和Septin-9(SEPT9)(DLS)。初步证据支持一种机制，在这种机制中，这种复合体将DCHS1- 通过其与细胞质LIX1L和SEPT9的相互作用，基于细胞与肌动蛋白细胞骨架的黏附。因此， 我们假设瓣膜重构是通过DCHS1-LIX1L-SEPT9-肌动蛋白机制发生的，这可能是 为MVP提供分子和细胞来源。这一假设将通过定义以下机制来检验： DLS复合体调节肌动蛋白的组织(目标1)，指导体外适当的瓣膜重塑(目标2)和 基因上在同一途径内相互作用，以调节适当的瓣膜几何形状和ECM组织(目标3)。 这项提案的目标1涉及一种体外方法来确定DLS对肌动蛋白细丝组织的影响 定量检测转基因小鼠细胞间隔蛋白-肌动蛋白网络的形成及由此产生的细胞内张力 心脏成纤维细胞。DLS与肌动蛋白细胞骨架相互作用的功能后果及其在 在AIM 2中，将通过应用一种新的体外方法来测量瓣膜重塑。 将从对照和全局DCHS1和/或LIX1L杂合子小鼠心脏中分离间质细胞(VIC) 并种植到3D生物工程瓣膜结构中，重塑天然瓣膜环境。读数 包括细胞排列、核形状、肌动蛋白组织、细胞外基质的产生和形成以及力量的产生 将被测量，并允许量化对阀门至关重要的重塑过程 形态发生。在目标3中进行的活体上位实验将增加每种方法的可信度，并将 确定DCHS1和Lix1L之间的遗传相互作用及其在我们提出的途径中的作用。这些研究 是重要的，因为它们是基于在MVP患者中发现的突变，并将定义分子和 世界上最常见的心血管疾病之一的细胞起源。

ABSTRACT Mitral valve prolapse (MVP) is one of the most common forms of cardiac valve disease and affects ~2-3% of the human population. There are no effective nonsurgical treatments for MVP and therapeutic efforts have been hindered by an incomplete understanding of its fundamental causes. However, we now have compelling genetic and functional evidence that significantly advances our understanding of MVP pathogenesis. Our group was the first to identify a genetic cause for MVP through identification of mutations in the atypical cadherin gene, DCHS1, in multiple families with non-syndromic MVP and have traced the origin of disease back to defects in fetal valve morphogenesis. The distinct functional and molecular consequences of DCHS1 deficiency are not currently known, but recent two-hybrid studies have revealed a novel protein complex between DCHS1, Lix-1 like (LIX1L), and Septin-9 (SEPT9) (DLS). Preliminary evidence supports a mechanism in which this complex links DCHS1- based cell adhesions to the actin cytoskeleton through its interactions with cytoplasmic LIX1L and SEPT9. Thus, we hypothesize that valve remodeling occurs through a DCHS1-LIX1L-SEPT9-actin mechanism, which may provide a molecular and cellular origin for MVP. This hypothesis will be tested by defining mechanisms by which the DLS complex regulates actin organization (Aim 1), directs proper valve remodeling ex vivo (Aim 2) and genetically interacts within the same pathway to regulate proper valve geometry and ECM organization (Aim 3). Aim 1 of this proposal involves an in vitro approach to define the effect of DLS on actin filament organization by quantifying septin-actin network formation and the resulting intracellular tension in genetically modified mouse cardiac fibroblasts. The functional consequences of DLS interactions with the actin cytoskeleton and its role in valve remodeling will be measured through application of a novel ex vivo approach in Aim 2. Here, valve interstitial cells (VICs) will be isolated from control and global Dchs1 and/or LIX1L heterozygote mouse hearts and seeded into a 3D bioengineered valve construct that recapitulates the native valve environment. Readouts including cell alignment, nuclear shape, actin organization, ECM production and formation, and force generation will be measured and allow for quantification of the remodeling processes that are crucial for valve morphogenesis. In vivo epistasis experiments performed in Aim 3 will add credence to each approach and will define the genetic interaction between Dchs1 and Lix1L and their role in our proposed pathway. These studies are significant since they are based on mutations identified in MVP patients and will define the molecular and cellular origins of one of the most common cardiovascular diseases in the world.