Larp6 and Alcoholic Cardiomyopathy

Larp6 和酒精性心肌病

基本信息

批准号：
10683154
负责人：
Joshua Edavettal
金额：
$ 5.27万
依托单位：
LSU HEALTH SCIENCES CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10683154
关键词：
Acceleration Adaptor Signaling Protein Affect Alcohol abuse Alcohol consumption Alcoholic Cardiomyopathy Alcohols Area Attenuated Binding Biological Markers Blood alcohol level measurement Cardiac Cardiac Catheterization Procedures Cardiology Cardiovascular Diseases Cells Chronic Clinical Collagen Data Development Disease Dose Echocardiography End Point Assay Ethanol Exposure to Family member Fibroblasts Fibrosis Functional disorder Health Heart Heart Hypertrophy Hour Hydroxyproline IL6 gene Immunohistochemistry In Vitro Inflammation Inflammatory Inflammatory Response Intervention LOX gene Left Length Lentivirus Vector Link Measures Mediator Mentors Messenger RNA Modeling Molecular Mus Mutate Myocardial dysfunction Myocarditis NF-kappa B National Institute on Alcohol Abuse and Alcoholism Phosphorylation Physicians Physiological Physiology Prevalence Process Proteins Rattus Research Research Personnel Ribonucleoproteins Role Scientist Series Side Stains Stress Structure Testing Training Transforming Growth Factor beta Translations United States United States National Institutes of Health Universities Ventricular Western Blotting Wild Type Mouse Work alcohol exposure alcohol response alcohol use disorder cardiogenesis chronic alcohol ingestion coronary fibrosis experimental study genetic manipulation heart damage heart function in vivo indexing interest mouse model novel novel therapeutics prevent receptor response small hairpin RNA small molecular inhibitor small molecule inhibitor therapeutic target

项目摘要

Abstract. Cardiac fibrosis and inflammation are hallmarks of alcoholic cardiomyopathy. A mediator of cardiac fibrosis, Larp6 interacts with the 5’ side loop (5’SL) of collagen I mRNA as an adapter protein which functions to upregulate collagen expression. In our studies, we target the interaction of Larp6 and collagen I mRNA using 1) genetic manipulation of Larp6 expression in cardiac fibroblasts, 2) a genetically modified mouse model, and 3) a novel small molecular inhibitor. End points include cardiac functional measures and inflammatory and fibrotic biomarkers in the heart and in isolated cardiac cells. First, we reduce Larp6 in cardiac fibroblasts (using shRNA delivered by a lentiviral vector) and use mutated cells and novel mice lacking functional 5’SL collagen I mRNA (5’SL mice). These interventions prevent Larp6 binding to collagen mRNA. In vivo exposure to alcohol follows the NIAAA chronic+binge model, and we compare the 5’SL mice to wildtype controls via serial echocardiography every two weeks until terminal, invasive left heart catheterization. Following extraction of the heart, we stain for collagen, marking fibrotic changes, while comparing left ventricular size to tibial length, as an index of cardiac hypertrophy. In vitro exposure to alcohol is up to 48 hours at various doses corresponding to blood alcohol concentration typical of clinical alcohol abuse. Using qPCR, Western blotting, and immunohistochemistry, we assess the following series of biomarkers in cardiac fibroblasts isolated from our mice: TRAF3IP2, IL1b, IL6, NFkB, and TNFa (to mark inflammation) and collagens I and III protein and mRNA, a-SMA, TGFβ, LOX and its activity, and hydroxyproline (to mark fibrosis). In addition to the genetic manipulation, we administer the novel small molecular inhibitor C9 - C9 inhibits the interaction of Larp6 and the 5’SL units of collagen I mRNA. The same cardiac functional measures (echocardiography and invasive catherization generating PV curves) are used for wildtype mice and endpoint assays of inflammatory and fibrotic markers are performed on cardiac fibroblasts. These data will establish the role of Larp6 in alcoholic cardiomyopathy, characterizing the cardiac fibroblast response to ethanol, and illustrating how Larp6 affects inflammatory, fibrotic, functional, and structural cardiac developments in alcoholic cardiomyopathy. Ultimately, these studies may identify novel, therapeutic targets for the treatment of alcoholic cardiomyopathy.

抽象的。心脏纤维化和炎症是酒精性心肌病的标志，是心脏纤维化的介质。 Larp6 作为衔接蛋白与 I 型胶原蛋白 mRNA 的 5' 侧环 (5'SL) 相互作用，其功能是在我们的研究中，我们使用 1) 来靶向 Larp6 和胶原蛋白 I mRNA 的相互作用。心脏成纤维细胞中 Larp6 表达的基因操作，2) 转基因小鼠模型，以及 3) 一种新型小分子抑制剂。终点包括心脏功能测量以及炎症和纤维化。心脏和分离的心肌细胞中的生物标志物。首先，我们减少心脏成纤维细胞中的 Larp6（使用慢病毒载体递送的 shRNA）并使用突变细胞以及缺乏功能性 5’SL 胶原蛋白 I mRNA 的新型小鼠（5’SL 小鼠）这些干预措施可阻止 Larp6 结合。体内暴露于酒精遵循 NIAAA 慢性+暴食模型，我们比较了每两周通过连续超声心动图将 5'SL 小鼠与野生型对照小鼠进行比较，直至终末、侵入性左心取出心脏后，我们对胶原蛋白进行染色，标记纤维化变化，同时将左心室大小与胫骨长度进行比较，作为体外暴露于酒精的指标。与临床酒精滥用典型的血液酒精浓度相对应的各种剂量可长达 48 小时。使用 qPCR、蛋白质印迹和免疫组织化学，我们评估了以下一系列生物标志物：从我们的小鼠中分离出的心脏成纤维细胞：TRAF3IP2、IL1b、IL6、NFkB 和 TNFa（用于标记炎症）和 I 型和 III 型胶原蛋白和 mRNA、a-SMA、TGFβ、LOX 及其活性，以及羟脯氨酸（用于标记纤维化）。除了基因操作之外，我们还施用新型小分子抑制剂 C9 - C9 抑制 Larp6 和 I 型胶原蛋白 mRNA 5’SL 单元的相互作用相同的心脏功能测量。（超声心动图和侵入性导管插入术生成 PV 曲线）用于野生型小鼠和终点对心脏成纤维细胞进行炎症和纤维化标记物的测定。这些数据将确定 Larp6 在酒精性心肌病中的作用，表征心脏成纤维细胞对乙醇的反应，并说明 Larp6 如何影响炎症、纤维化、功能性和结构性心脏最终，这些研究可能会确定新的治疗靶点。酒精性心肌病的治疗。