Mouse Models of Non-Syndromic Cardiac Progeria

描述（由申请方提供）：目的是开发和表征非综合征性心脏早老症（NSCP）的遗传小鼠模型。这些研究的动力来自NSCP在患有过早和进行性退行性心血管疾病的年轻患者中的发现，这是衰老的特征。临床和遗传学研究使我们提倡这样一种学说，即心脏参与LMNA（核纤层蛋白A/C）突变，称为心肌病代表NSCP。我们建议开发和表征小鼠模型表达突变LmnaD 300 N和LmnaR 644 C发现在患者进行性退行性心血管疾病。p.D300N与涉及心肌、传导系统、瓣膜和冠状动脉的退行性疾病相关，而p.R644C是心肌病的最常见原因。 我们将使用重组工程（重组介导的基因工程）方法来产生LmnaD 300 N、LmnaR 644 C和Lmna-野生型（WT）转基因小鼠，后者作为对照。基因工程改造是一种相对新颖和强大的方法，其能够有效构建除预期突变/变化之外与WT基因相似的转基因载体。该方法利用E.大肠杆菌中使用从λ噬菌体提供的重组蛋白。将重组的转基因构建体显微注射到受精的单细胞小鼠胚胎中以产生遗传小鼠模型，其中转基因的表达受支配内源基因表达的调节序列调节。这些模型将在遗传上概括Lmna突变的人类基因型，并且还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。 衰老的表型在分子水平上通过诱导表达已建立的衰老标志物来定义，即衰老相关的2-半乳糖苷酶（S-A 2-Gal）、p16 INK 4a（Cdkn 2a）、P19、p21 WAF 1/CIP 1、端粒磨损和端粒酶活性。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;通过超声心动图评估心脏结构和功能;通过心电图评估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（TGF 2）的营养（肌细胞），增殖和修复（成纤维细胞）反应。此外，我们将定量端粒酶活性，端粒磨损和p16 INK 4a，p21 WAF 1/CIP 1，SA-2-Gal和氧化应激标记物在心脏中的水平。这些研究有望建立NSCP小鼠模型，并为阐明心脏衰老的基本机制提供机会。这一发现有助于预防、减轻和治愈人类的心肌病。 公共卫生相关性：在患有早发性退行性心血管疾病的人类患者中进行的临床和遗传研究，衰老的特征，使我们倡导LMNA（核纤层蛋白A/C）突变引起非综合征性心脏早衰症的学说。为了验证这一假设，我们建议开发和表征与非综合征性心脏早衰症相关的特定LMNA突变的重组小鼠模型。该模型为阐明心肌病的分子和细胞机制提供了机会，并为揭示心血管衰老的机制提供了线索。

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.