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.

摘要 二尖瓣脱垂（MVP）是心脏瓣膜疾病最常见的形式之一，影响约2-3%的患者。 人口。MVP没有有效的非手术治疗方法， 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， 和功能性证据，显著推进了我们对MVP发病机制的理解。我们组是 首先通过鉴定非典型钙粘蛋白基因DCHS 1的突变来鉴定MVP的遗传原因， 在多个非综合征型MVP家族中， 形态发生DCHS 1缺陷的不同功能和分子后果目前还不清楚。 已知的，但最近的双杂交研究已经揭示了DCHS 1，Lix-1样（LIX 1 L）， 和Septin-9（SEPT 9）（DLS）。初步证据支持这种复合物将DCHS 1- 通过与细胞质LIX 1 L和SEPT 9的相互作用，基于细胞粘附到肌动蛋白细胞骨架。因此，在本发明中， 我们假设瓣膜重塑是通过DCHS 1-LIX 1 L-SEPT 9-actin机制发生的， 为MVP提供分子和细胞来源。这一假设将通过定义机制来检验， DLS复合物调节肌动蛋白组织（Aim 1），指导离体适当的瓣膜重塑（Aim 2）， 在相同的通路内遗传相互作用，以调节适当的瓣膜几何形状和ECM组织（目的3）。 本提案的目的1涉及一种体外方法，通过以下方法来确定DLS对肌动蛋白丝组织的影响： 在遗传修饰的小鼠中定量septin-actin网络形成和产生的细胞内张力 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，将通过应用新的离体方法测量瓣膜重塑。这里，阀门 将从对照和整体Dchs 1和/或LIX 1 L杂合子小鼠心脏中分离间质细胞（VIC） 并接种到重现天然瓣膜环境的3D生物工程瓣膜构造中。读数 包括细胞排列、核形状、肌动蛋白组织、ECM产生和形成以及力的产生 将被测量，并允许量化对瓣膜至关重要的重塑过程 形态发生在目标3中进行的体内上位性实验将增加每种方法的可信度， 定义Dchs 1和Lix 1 L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MVP患者中鉴定的突变， 世界上最常见的心血管疾病之一的细胞起源。