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

Dchs1 和 Septin 细胞骨架：二尖瓣脱垂的分子和发育病因学

• 批准号: 10383138
• 负责人: Kelsey Schuyler Moore
• 金额: $ 1.33万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2021-07-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10383138
• 关键词: 3-Dimensional; Actins; Adult; Affect; Arrhythmia; Back; Biological Assay; Biological Models; Biomedical Engineering; Cadherins; Cardiac; Cardiac Surgery procedures; Cardiovascular Diseases; Cardiovascular system; Cell Adhesion; Cell Nucleus; Cells; Collagen; Complex; Cytoskeleton; DNA; DNA Sequence Alteration; Defect; Development; Disease; Environment; Etiology; Extracellular Matrix; F-Actin; Family; Fibrin; Fibroblasts; Foundations; Future; Generations; Genes; Genetic; Genetic Epistasis; Geometry; Goals; Health; Heart; Heart Valve Diseases; Heart failure; Heterozygote; Histologic; Human; Human Genetics; In Vitro; Intervention; Lead; Link; Measurement; Measures; Membrane; Methods; Microfilaments; Mitral Valve; Mitral Valve Prolapse; Modeling; Molecular; Morbidity - disease rate; Morphogenesis; Mus; Mutation; Nuclear; Operative Surgical Procedures; Pathogenesis; Pathway interactions; Patients; Peptides; Pharmacology; Phase; Phenocopy; Population; Pregnancy; Process; Production; Role; Secondary to; Seeds; Series; Shapes; Structure; System; Testing; Therapeutic; Thinness; Tissues; Transmission Electron Microscopy; Western World; base; clinically relevant; experimental study; fetal; immunocytochemistry; in vivo; insight; interstitial cell; mortality; mouse model; novel; novel therapeutics; polymerization; postnatal; protein complex; sudden cardiac death; three-dimensional modeling; treatment strategy; yeast two hybrid system

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.

抽象的 二尖瓣脱垂（MVP）是心脏瓣膜疾病的最常见形式之一，影响约2-3％ 人口。对于MVP和治疗努力，没有有效的非手术治疗方法 受到对其基本原因的不完全理解的阻碍。但是，我们现在有引人入胜的遗传 以及显着提高我们对MVP发病机理的功能证据。我们的小组是 首先通过鉴定非典型钙粘蛋白基因DCHS1中的突变来识别MVP的遗传原因 在多个非综合征MVP的家族中，并将疾病的起源追溯到胎瓣缺陷 形态发生。 DCHS1缺乏的独特功能和分子后果目前不是 已知的，但最近的两种杂交研究揭示了DCHS1，Lix-1（lix1l）之间的新型蛋白质复合物， 和9月9日（9月9日）（DLS）。初步证据支持了一种机制，在这种机制中，这种复合物将DCHS1-联系起来 通过与细胞质LIX1L和SEPT9的相互作用，基于肌动蛋白细胞骨架的细胞粘附。因此， 我们假设阀重塑是通过DCHS1-LIX1L-SEPT9-ACTIN机制进行的，该机制可能 为MVP提供分子和细胞来源。该假设将通过定义机制来检验 DLS综合体调节肌动蛋白组织（AIM 1），指导适当的阀重塑离体（AIM 2）和 基因在相同的途径中相互作用，以调节适当的阀几何形状和ECM组织（AIM 3）。 该提案的目标1涉及一种体外方法，以定义DLS对肌动蛋白细丝组织的影响 量化septin-actin网络形成和基因修饰小鼠的细胞内张力 心脏成纤维细胞。 DLS与肌动蛋白细胞骨架的功能后果及其在 阀重塑将通过在AIM 2中应用新型的离体方法来测量。在这里，阀门 间质细胞（VIC）将从对照和全局DCHS1和/或Lix1l杂合小鼠心脏中分离出来 并播种成3D生物工程的阀构建体，该阀构建了本地阀环境。读数 包括细胞对齐，核形状，肌动蛋白组织，ECM生产和形成以及力产生 将测量并允许量化对阀门至关重要的重塑过程 形态发生。在AIM 3中执行的体内上位性实验将为每种方法增加可信度，并将 定义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 很重要，因为它们基于在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。