Larp6 and Alcoholic Cardiomyopathy

Larp6 和酒精性心肌病

• 批准号: 10537563
• 负责人: Joshua Edavettal
• 金额: $ 5.18万
• 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/10537563
• 关键词: Adaptor Signaling Protein; Affect; Agonist; Alcohol abuse; Alcohol consumption; Alcoholic Cardiomyopathy; Alcohols; Area; Attenuated; Binding; Biological Markers; Blood alcohol level measurement; Cardiac; Cardiac Catheterization Procedures; Cardiac development; 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; Lead; Left; Length; Lentivirus Vector; Link; Measures; Mediator of activation protein; Mentors; Messenger RNA; Modeling; Molecular; Mus; Mutate; Myocardial dysfunction; Myocarditis; National Institute on Alcohol Abuse and Alcoholism; 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; base; chronic alcohol ingestion; coronary fibrosis; experimental study; genetic manipulation; heart damage; heart function; in vivo; indexing; interest; mouse model; new therapeutic target; novel; novel therapeutics; prevent; receptor; response; small hairpin RNA; small molecular inhibitor; small molecule inhibitor

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。心脏纤维化的介质， Larp 6作为衔接蛋白与胶原I mRNA的5'侧环（5' SL）相互作用，其功能是 上调胶原蛋白表达。在我们的研究中，我们使用以下方法靶向Larp 6和胶原I mRNA的相互作用：1） 心脏成纤维细胞中Larp 6表达的遗传操作，2）遗传修饰的小鼠模型，和3） 一种新型小分子抑制剂。终点包括心脏功能测量和炎症和纤维化 心脏和分离的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Larp 6（使用慢病毒载体递送的shRNA），并使用突变的细胞 和缺乏功能性5 ′ SL胶原I mRNA的新型小鼠（5 ′ SL小鼠）。这些干预措施阻止Larp 6结合 到胶原mRNA。体内暴露于酒精遵循NIAAA慢性+狂欢模型，我们比较了 5 'SL小鼠通过连续超声心动图每两周一次至野生型对照，直至终末，侵入性左心 漂浮导管心脏取出后，我们对胶原蛋白进行染色，标记纤维化变化， 比较左心室大小与胫骨长度，作为心脏肥大的指标。体外暴露于酒精是 在对应于临床酒精滥用典型的血液酒精浓度的各种剂量下长达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的心脏成纤维细胞：TRAF 3 IP 2、IL 1b、IL 6、NFkB和TNF α（标记炎症）， 胶原I和III蛋白和mRNA、α-SMA、TGFβ、LOX及其活性和羟脯氨酸（用于标记纤维化）。 除了遗传操作，我们还给予新的小分子抑制剂C9 - C9抑制了 Larp 6与I型胶原mRNA的5 'SL单位的相互作用。相同的心功能指标 （超声心动图和侵入性导管插入术产生PV曲线）用于野生型小鼠和终点 对心脏成纤维细胞进行炎性和纤维化标记物的测定。 这些数据将确定Larp 6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Larp 6如何影响炎症，纤维化，功能性和结构性心脏 酒精性心肌病的进展最终，这些研究可能会发现新的治疗靶点， 酒精性心肌病的治疗