Mechanistic Basis of Cardiac Laminopathy

项目摘要 核纤层蛋白A/C基因（LMNA）编码核纤层结构蛋白的突变负责 高达百分之十的遗传性扩张型心肌病这种疾病通常被称为心脏病 核纤层蛋白病实验证据部分支持各种致病机制的缺陷如何在核 结构蛋白引起心肌病，包括它们导致细胞机械稳定性异常， 基因表达失调和改变的细胞信号传导。然而，没有统一的假设来整合 这些有缺陷的过程，并准确地解释他们如何导致心肌细胞损伤和功能障碍。我们 最近发现异常细胞外信号调节激酶1/2（ERK 1/2） 信号传导和改变的核定位。这让我们假设 一个机械检查点，其中核纤层的改变上调ERK 1/2活性，这导致 通过磷酸化和失活肌动蛋白成束活性的细胞核的错位 含同源结构域蛋白（FHOD）。FHOD的失活阻止了核骨架的连接子， 细胞骨架（LINC）复合物，跨越核膜内外并连接到肌动蛋白 丝，以介导核定位。通常情况下，机械检查站的作用是防止过度用力 在收缩心肌细胞时被应用于细胞核。然而，随着核武器的永久性改变， 由于LMNA突变导致的结构，持续激活的检查点变得适应不良， 核定位异常、核膜破裂、DNA损伤和肌节功能缺陷。这 项目旨在证明核机械检查点假说，并确定其在 心肌纤层病的发病机制。在目标1中，我们将研究如何激活机械检查点， 核定位改变心肌细胞生物学。我们将直接测量作用在原子核上的力- 2肌动蛋白张力传感器。由于最近的数据表明，细胞核有助于正常的肌节，我们将测试 假设持续的机械检查点激活和核错位导致缺陷的 肌节组装和功能。在目标3中，我们将确定如何改变机械 检查点在体内影响心脏。我们将测试是否表达拟磷酸化FOHD变体（检查点 激活）在野生型小鼠心脏中诱导心肌病， （检查点失活）改善了心脏纤层蛋白病小鼠模型中的病理学。证明了 一种新的核机械检查点，并建立其在心肌病发病机制中的作用， LMNA突变将改变该领域的研究方向，并可能导致新的治疗方法- 威胁遗传性心脏病

PROJECT SUMMARY Mutations in the lamin A/C gene (LMNA) encoding structural proteins of the nuclear lamina are responsible for up to ten percent of cases of inherited dilated cardiomyopathy. The disease is often referred to as cardiac laminopathy. Experimental evidence partially supports various pathogenic mechanisms of how defects in nuclear structural proteins cause cardiomyopathy, including that they lead to abnormalities in cell mechanical stability, dysregulation of gene expression and altered cell signaling. However, there is no unifying hypothesis integrating these defective processes and explaining exactly how they lead to cardiomyocyte damage and dysfunction. We recently found a surprising relationship between aberrant extracellular signal-regulated kinase 1/2 (ERK1/2) signaling and altered nuclear positioning in cardiac laminopathy. This has led us to hypothesize the existence of a mechanic checkpoint in which alterations in the nuclear lamina upregulate ERK1/2 activity, which causes mispositioning of the nucleus by phosphorylating and inactivating the actin bundling activity of the formin homology domain-containing protein (FHOD). Inactivation of FHOD prevents the linker of nucleoskeleton and cytoskeleton (LINC) complex, which spans the inner and outer nuclear membranes and connects to actin filaments, to mediate nuclear positioning. Normally, the mechanical checkpoint acts to prevent excessive force from being applied to the nucleus in contracting cardiomyocytes. However, with permanent alterations in nuclear structure resulting from LMNA mutations, the persistently activated checkpoint becomes maladaptive, resulting in abnormal nuclear positioning, nuclear envelope rupture, DNA damage and defects in sarcomere function. This Project is designed to prove the nuclear mechanical checkpoint hypothesis and determine its role in the pathogenesis of cardiac laminopathy. In Aim 1, we will examine how activation of the mechanical checkpoint for nuclear positioning alters cardiomyocyte biology. We will directly measure force on the nucleus using a nesprin- 2 actin tension sensor. As recent data suggest that the nucleus contributes to normal sarcomere, we will test the hypothesis that persistent mechanical checkpoint activation and nuclear mispositioning leads to defective sarcomere assembly and function in cardiomyocytes. In Aim 3, we will determine how altering the mechanical checkpoint affects the heart in vivo. We will test if expressing a phosphomimetic FOHD variant (checkpoint activation) in the heart induces cardiomyopathy in wild type mouse hearts and if a non-phosphorylatable variant (checkpoint inactivation) ameliorates pathology in a mouse model of cardiac laminopathy. Proving the existence of a novel nuclear mechanical checkpoint and establishing its role in the pathogenesis of cardiomyopathy caused by LMNA mutations will shift research directions in the field and potentially lead to new treatments for this life- threatening inherited heart disease.