Mechanistic Basis of Cardiac Laminopathy

项目总结 编码核层结构蛋白的层蛋白A/C基因(LMNA)突变是导致 高达10%的遗传性扩张型心肌病患者。这种疾病通常被称为心脏病。 椎板病。实验证据部分支持不同的致病机制，即核缺陷 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改变。然而，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是如何导致心肌细胞损伤和功能障碍的。我们 最近发现异常的细胞外信号调节激酶1/2(ERK1/2)之间存在令人惊讶的关系 心脏椎板病中的信号转导和核位置改变。这使我们假设存在 一种机械检查点，核板的变化会上调ERK1/2的活性，从而导致 通过磷酸化和失活形成蛋白的肌动蛋白束活性导致核的错误定位 同源结构域包含蛋白(FHOD)。FHOD的失活阻止了核骨架的连接子和 细胞骨架(LINC)复合体，横跨内、外核膜，与肌动蛋白相连 细丝，以调节核定位。通常情况下，机械检查站的作用是防止过度使用武力 在收缩心肌细胞的过程中应用于细胞核。然而，随着原子核的永久性变化 结构，持续激活的检查点变得不适应，导致 核定位异常、核膜破裂、DNA损伤和肌节功能缺陷。这 该项目旨在证明核机械检查点假说，并确定其在 心脏椎板病的发病机制。在目标1中，我们将研究机械检查点的激活如何 核定位改变了心肌细胞的生物学。我们将使用内斯普林直接测量原子核上的力- 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定位错误会导致缺陷 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 检查点在活体内影响心脏。我们将测试是否表达仿磷FOHD变异体(检查点 激活)在野生型小鼠心脏中诱导心肌病，如果是非磷酸化变异体 (检查点失活)改善心脏椎板病小鼠模型的病理。证明它的存在 一种新的核机械检查点及其在心肌病发病机制中的作用 通过LMNA突变将改变该领域的研究方向，并可能导致针对这一生命的新疗法- 威胁到遗传性心脏病。

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.