Larp6 and Alcoholic Cardiomyopathy

Larp6 和酒精性心肌病

• 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
• 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

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。心脏纤维化介质， LARP6与胶原蛋白I mRNA的5'侧环（5’sl）作为衔接蛋白相互作用 上调胶原蛋白表达。在我们的研究中，我们使用1靶向LARP6和胶原蛋白I mRNA的相互作用。 心脏成纤维细胞中LARP6表达的遗传操纵，2）转基因的小鼠模型和3） 一种新型的小分子抑制剂。终点包括心脏功能测量以及炎症和纤维化 心脏和孤立心脏细胞中的生物标志物。 首先，我们减少了心脏成纤维细胞中的LARP6（使用慢病毒载体传递的shRNA）并使用突变的细胞 还有缺乏功能性5'sl胶原蛋白mRNA（5'SL小鼠）的新型小鼠。这些干预措施阻止了LARP6结合 到胶原蛋白mRNA。体内暴露于酒精的遵循NIAAA慢性+暴饮暴食模型，我们比较 5'SL小鼠通过连续超声心动图进行一次野外型对照，直到终端，侵入性左心 导管插入。提取心脏后，我们染色胶原蛋白，标志着纤维化的变化，而 将左心室大小与胫骨长度进行比较，作为心脏肥大的指数。体外暴露于酒精是 多种剂量的最多48小时，对应于典型的临床酒精滥用的血液酒精浓度。 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的生物标志物 与我们的小鼠分离的心脏成纤维细胞：TRAF3IP2，IL1B，IL6，NFKB和TNFA（标记炎症）和 胶原I和III蛋白和mRNA，A-SMA，TGFβ，LOX及其活性以及羟基丙烯酸酯（标记纤维化）。 除了遗传操作外，我们还管理新型的小分子抑制剂C9 -C9抑制了 LARP6和胶原蛋白I mRNA的5'SL单位的相互作用。相同的心脏功能测量 （超声心动图和侵入性收缩化生成PV曲线）用于野生型小鼠和端点 对心脏成纤维细胞进行炎症和纤维化标记的测定。 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，功能和结构心脏 酒精性心肌病的发展。最终，这些研究可能会确定新颖的治疗目标 酒精性心肌病的治疗。