Mechanistic Basis of Cardiac Laminopathy

项目概要 编码核纤层结构蛋白的核纤层蛋白 A/C 基因 (LMNA) 的突变负责 高达百分之十的遗传性扩张型心肌病病例。这种疾病通常被称为心脏病 椎板病。实验证据部分支持核缺陷如何产生的各种致病机制 结构蛋白导致心肌病，包括它们导致细胞机械稳定性异常， 基因表达失调和细胞信号传导改变。然而，并没有统一的假设来整合 这些有缺陷的过程并准确解释它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 (ERK1/2) 之间存在令人惊讶的关系 心脏纤层病中的信号传导和核定位改变。这使我们推测存在 一个机械检查点，其中核纤层的改变上调 ERK1/2 活性，从而导致 通过磷酸化和灭活福尔明的肌动蛋白成束活性而导致细胞核错位 含同源结构域的蛋白（FHOD）。 FHOD 失活会阻止核骨架的连接 细胞骨架 (LINC) 复合物，跨越内核膜和外核膜并与肌动蛋白连接 细丝，介导核定位。通常，机械检查点的作用是防止用力过大 应用于收缩心肌细胞的细胞核。然而，随着核的永久改变 LMNA 突变产生的结构，持续激活的检查点变得适应不良，导致 核定位异常、核膜破裂、DNA 损伤和肌节功能缺陷。这个 该项目旨在证明核机械检查点假说并确定其在核机械检查点假说中的作用 心脏纤层病的发病机制。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。我们将使用 Nesprin 直接测量细胞核上的力 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 心肌细胞中肌节的组装和功能。在目标 3 中，我们将确定如何改变机械 检查点影响体内的心脏。我们将测试是否表达拟磷FOHD变体（检查点 心脏中的激活）会在野生型小鼠心脏中诱导心肌病，并且如果非磷酸化变体 （检查点失活）改善小鼠心脏纤层病模型的病理学。证明存在性 一种新型核机械检查点的发现及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，并可能带来新的治疗方法—— 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.