Ryanodine Receptor Defects in Cardiomyopathy Caused by Lamin A/C Gene Mutations

项目摘要 由编码A型核纤层蛋白的核纤层蛋白A/C基因（LMNA）突变引起的扩张型心肌病， 一种无法治愈的致命疾病心肌病的致病机制 对这种遗传性疾病的了解很少。特别是，目前尚不清楚蛋白质的改变是如何发生的。 在几乎所有终末分化细胞的细胞核中表达，选择性地导致心脏病。我们的假设 A型核纤层蛋白的改变使细胞易受氧化应激诱导的兰尼定重塑的影响 受体（RyR），产生肌浆网（SR）Ca 2+“泄漏”。氧化应激和细胞溶质增加 Ca 2+也有助于细胞外信号调节激酶1和2（ERK 1/2）的过度激活， LMNA突变引起的心肌病。细胞内Ca ~（2+）和ERK_（1/2）活性升高 产生各种缺陷，包括线粒体功能障碍，导致心肌病。我们的一个推论 一种假说认为，阻断SR Ca 2+“泄漏”对LMNA引起的心肌病有有益作用 突变。我们将使用该疾病的小鼠模型和人体组织来检验我们的假设及其推论。 在目标1中，我们将确定导致心肌病的A型核纤层蛋白改变是否会导致心脏功能增强 肌肉氧化应激，导致RyR 2重塑和SR Ca 2+“泄漏”。我们还将确定SR是否 Ca 2+“泄漏”刺激ERK 1/2活性，导致线粒体功能障碍和DNA损伤。除了 心脏，我们将类似地检查骨骼肌，这通常同时影响人类患者， LMNA突变引起的心肌病以及模型小鼠。我们将进一步评估这些过程 在稳定表达引起心肌病的核纤层蛋白A变体或缺乏A型核纤层蛋白的培养细胞中。在目标2中， 我们将利用RyR的三维结构来确定如何进行特定的氧化修饰， Lmna突变小鼠横纹肌内Ca 2+浓度的增加影响了横纹肌的结构，使横纹肌内Ca 2+“渗漏”。在目标3中，我们 进行实验以确定Rycal，稳定重构RyR并阻断SR Ca 2+的药物， “泄漏”，改善Lmna突变小鼠的心脏功能和心脏存活率， 来自患有由LMNA突变引起的心肌病的人类受试者。我们将进一步确定Rycal是否有 当与ERK 1/2活性的抑制剂组合时，其具有协同的有益效果， 在患有心肌病的Lmna突变小鼠中显示出部分改善心脏功能。这些研究将揭示 关于LMNA突变引起的心肌病发病机制的基本信息， 核内蛋白缺陷与心功能不全的有形机制。他们还将确定药物是否 已经在临床开发中，可以转化为对这种致命心脏病患者的试验。

Project Summary Dilated cardiomyopathy caused by mutations in the lamin A/C gene (LMNA) encoding A-type nuclear lamins is a life-threatening disease with no definitive cure. The pathogenic mechanisms responsible for cardiomyopathy in this inherited disease are poorly understood. In particular, it is not known how alterations in proteins expressed in nuclei of virtually all terminally differentiated cells selectively cause heart disease. Our hypothesis is that alterations in A-type lamins predispose cells to oxidative stress-induced remodeling of ryanodine receptors (RyRs), creating a sarcoplasmic reticulum (SR) Ca2+ “leak.” Oxidative stress and increased cytosolic Ca2+ also contribute to hyper-activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which occurs in cardiomyopathy caused by LMNA mutations. The increased cytosolic Ca2+ and ERK1/2 activity generates various defects, including mitochondrial dysfunction, that cause cardiomyopathy. A corollary of our hypothesis is that blocking the SR Ca2+ “leak” will have beneficial effects in cardiomyopathy caused by LMNA mutations. Using mouse models of the disease and human tissue, we will test our hypothesis and its corollary. In Aim 1, we will determine if alterations in A-type lamins that cause cardiomyopathy lead to enhanced cardiac muscle oxidative stress, resultant RyR2 remodeling and a SR Ca2+ “leak.” We will also determine if the SR Ca2+ “leak” stimulates ERK1/2 activity, causes mitochondrial dysfunction and damages DNA. In addition to heart, we will similarly examine skeletal muscle, which is often simultaneously affected in human patients with cardiomyopathy caused by LMNA mutations as well as in model mice. We will further assess these processes in cultured cells that stably express a cardiomyopathy-causing lamin A variant or lack A-type lamins. In Aim 2, we will utilize the three-dimensional structure of RyR to determine how specific oxidative modifications that occur in striated muscle of Lmna mutant mice affect its structure and make it “leaky” to Ca2+. In Aim 3, we will perform experiments to determine if a Rycal, drugs that stabilize remodeled RyRs and block the SR Ca2+ “leak,” improves heart function and prolongs survival in Lmna mutant mice and if it blocks the “leak” in hearts from human subjects with cardiomyopathy caused by LMNA mutations. We will further determine if a Rycal has synergistically beneficial effects when combined with an inhibitor of ERK1/2 activity, which has previously been shown to partially improve heart function in Lmna mutant mice with cardiomyopathy. These studies will reveal basic information about the pathogenesis of cardiomyopathy caused by LMNA mutations and connect an intranuclear protein defect with a tangible mechanism of cardiac dysfunction. They will also determine if drugs already in clinical development can be translated to trials in patients with this lethal heart disease.