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

项目摘要 编码A型核蛋白的层蛋白A/C基因(LMNA)突变引起的扩张型心肌病 这是一种威胁生命的疾病，目前尚无确切的治疗方法。心肌病的致病机制 人们对这种遗传性疾病知之甚少。特别是，目前还不知道蛋白质的变化是如何 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型板层的改变使细胞容易受到氧化应激诱导的兰尼定重塑 受体(RyRs)，造成肌浆网(SR)钙“泄漏”。氧化应激与胞浆升高 CA2还促进细胞外信号调节激酶1和2(ERK1/2)的过度激活，ERK1/2 发生在由LMNA突变引起的心肌病中。细胞内钙离子和ERK1/2活性升高 产生各种缺陷，包括线粒体功能障碍，导致心肌病。这是我们的推论 假说认为阻断肌浆网钙“泄漏”对LMNA所致心肌病有有利作用 突变。使用疾病的小鼠模型和人体组织，我们将检验我们的假设及其推论。 在目标1中，我们将确定引起心肌病的A型板层的改变是否会导致心脏功能增强 肌肉氧化应激，导致RyR2重塑和肌浆网钙离子“泄漏”。我们还将确定SR是否 CA2“泄漏”刺激ERK1/2活性，导致线粒体功能障碍和DNA损伤。除了……之外 心脏，我们将类似地检查骨骼肌，它经常同时受到影响的人类患者 由LMNA突变引起的心肌病以及在模型小鼠中。我们将进一步评估这些进程 在稳定表达导致心肌病的层粘连蛋白A变体或缺乏A型粘连蛋白的培养细胞中。在目标2中， 我们将利用RyR的三维结构来确定特定的氧化修饰如何 在Lmna突变小鼠的横纹肌中发生的钙离子会影响其结构，并使其对钙离子“泄漏”。在《目标3》中，我们将 进行实验以确定Rycal，一种稳定重塑的RyRs并阻断SR钙的药物 “漏”可改善Lmna突变小鼠的心功能并延长其存活时间，如果它能阻断心脏的“漏” 来自由LMNA突变引起的心肌病患者。我们将进一步确定Rycal是否有 与ERK1/2活性抑制剂联合使用时的协同有益效应，此前已有 显示能部分改善患有心肌病的LmNA突变小鼠的心功能。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及其与 核内蛋白缺陷，具有明显的心脏功能障碍机制。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.