Mouse Models of Non-Syndromic Cardiac Progeria

描述（由申请人提供）：目的是开发和表征非综合征性心源性早衰（NSCP）的遗传小鼠模型。这项研究的动力来自于在患有早衰和进行性退行性心血管疾病的年轻患者中发现的NSCP，这是衰老的特征。临床和遗传学研究使我们主张心脏参与LMNA （Lamin A/C）突变，即心氨病代表NSCP的学说。我们建议在进行性退行性心血管疾病患者中建立表达突变体LmnaD300N和LmnaR644C的小鼠模型并对其进行表征。p.D300N与涉及心肌、传导系统、瓣膜和冠状动脉的退行性疾病有关，而p.R644C是心肌病的最常见原因。我们将使用重组（重组介导的基因工程）方法产生LmnaD300N， LmnaR644C和lmna野生型（WT），后者作为对照，转基因小鼠。重组是一种相对新颖和强大的方法，可以有效地构建与WT基因相似的转基因载体，除了预期的突变/改变。该方法利用来自lambda噬菌体的重组蛋白在大肠杆菌中进行同源重组。将重组的转基因构建物微注射到受精卵单细胞小鼠胚胎中，产生转基因小鼠模型，其中转基因的表达受内源基因表达的调控序列的调控。这些模型将从基因上概括人类基因型的Lmna突变，也将能够分析多种心脏和非心脏细胞类型的参与，包括肌细胞、成纤维细胞和传导系统。衰老的表型是在分子水平上通过诱导表达衰老相关的2-半乳糖苷酶（S-A 2-Gal）、p16INK4a （Cdkn2a）、P19、p21WAF1/CIP1、端粒磨损和端粒酶活性来定义的。在细胞水平上，我们将评估对激动剂的营养、复制和修复反应，在器官水平上评估心肌功能障碍和传导缺陷。我们将通过Kaplan-Meier生存分析评估生存率；超声心动图检查心脏结构和功能；通过心电图检查传导缺陷和心律失常。同样，我们将分析从心脏分离的细胞对血管紧张素II （AT）和转化生长因子2 （TGF2）的营养（肌细胞）、增殖和修复（成纤维细胞）反应。此外，我们将量化端粒酶活性、端粒磨损以及心脏中p16INK4a、p21WAF1/CIP1、SA-2-Gal和氧化应激标志物的水平。这些研究有望建立NSCP小鼠模型，并为阐明控制心脏衰老的基本机制提供机会。这些发现有助于预防、减轻和治疗人类心肌病。

DESCRIPTION (provided by applicant): The objective is to develop and characterize genetic mouse models of non-syndromic cardiac progeria (NSCP). The impetus for the studies is originated from the findings of NSCP in young patients with premature and progressive degenerative cardiovascular disease, characteristic of aging. Clinical and genetic studies have led us to advocate the doctrine that cardiac involvement in LMNA (Lamin A/C) mutations, referred to as cardiolaminopathy represents NSCP. We propose to develop and characterize mouse models expressing mutant LmnaD300N and LmnaR644C found in patients with progressive degenerative cardiovascular diseases. The p.D300N is associated with degenerative disease involving myocardium, conduction system, valves and the coronary arteries, while the p.R644C is the most common cause of cardiolaminopathy. We will use the recombineering (recombination-mediated genetic engineering) method to generate LmnaD300N, LmnaR644C and Lmna-wild type (WT), latter as a control, transgenic mice. Recombineering is a relatively novel and powerful method that enables efficient construction of the transgene vectors that are similar to the WT gene with the exception of the intended mutation/changes. The method utilizes homologous recombination in E. coli using recombination proteins provided from a lambda phage. The recombined transgene constructs are microinjected into fertilized single cell mouse embryos to produce genetic mouse models in which expression of the transgene is regulated by the regulatory sequences that govern expression of the endogenous gene. The models will genetically recapitulate the human genotype of Lmna mutations and also will enable analyzing involvement of multiple cardiac and non-cardiac cell types, including myocytes, fibroblasts and conduction system. The phenotype of aging is defined at the molecular level by induced expression of established markers of senescence, namely senescence-associated 2-galactosidase (S-A 2-Gal), p16INK4a (Cdkn2a), P19, p21WAF1/CIP1, telomere attrition and telomerase activity. At the cellular level we will assess trophic, replicative and reparative responses to agonists and at the organ level myocardial dysfunction and conduction defects. We will assess survival by Kaplan-Meier survival analysis; cardiac structure and function by echocardiography; and conduction defects and arrhythmias by electrocardiography. Likewise, we will analyze trophic (myocyte), proliferative and reparative (fibroblasts) responses of cells isolated from the hearts to angiotensin II (AT) and transforming growth factor 2 (TGF2). Moreover, we will quantify telomerase activity, telomere attrition and levels of p16INK4a, p21WAF1/CIP1, SA-2-Gal and markers of oxidative stress in the heart cardiac. The studies are expected to establish the proposed models as mouse models of NSCP and provide the opportunity to elucidate the fundamental mechanisms that govern cardiac senescence. The discoveries could facilitate prevention, attenuation and cure of cardiolaminopathy in humans. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Clinical and genetic studies in human patients with premature degenerative cardiovascular diseases, characteristics of aging, have led us to advocate the doctrine that LMNA (Lamin A/C) mutations cause non- syndromic cardiac progeria. To test this hypothesis we propose to develop and characterize recombineered mouse models of specific LMNA mutations that are associated with the non-syndromic cardiac progeria. The models could afford the opportunity to delineate the molecular and cellular mechanisms of cardiolaminopathy and shed light into the mechanisms of cardiovascular senescence.