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。 心肌纤维化和炎症是酒精性心肌病的特征。心脏纤维化的中介物， Larp6与I型胶原mRNA的5‘侧环(5’SL)相互作用，作为一种适配蛋白发挥作用 上调胶原蛋白表达。在我们的研究中，我们使用1)来靶向Larp6和I型胶原mRNA的相互作用 心脏成纤维细胞中Larp6表达的遗传操作，2)转基因小鼠模型，3) 一种新型小分子缓蚀剂。终点包括心功能指标以及炎症和纤维化。 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)，并使用突变细胞 和缺乏功能性5‘SL胶原蛋白I mRNA的新小鼠(5’SL小鼠)。这些干预阻止了Larp6的结合 胶原蛋白基因的表达。在体内接触酒精遵循NIAAA慢性+酗酒模型，我们比较了 5‘SL小鼠至野生型对照，每隔两周进行一次连续超声心动图检查，直至末梢，有创左心 导尿术。心脏摘除后，我们对胶原蛋白进行染色，以标记纤维化的变化，而 比较左心室大小和胫骨长度，作为心肌肥厚的指标。在体外暴露于酒精是 根据临床酒精滥用的典型血液酒精浓度，长达48小时的不同剂量。 使用qPCR、Western blotting和免疫组织化学方法，我们评估了以下一系列生物标记物在 从我们的小鼠分离的心脏成纤维细胞：TRAF3IP2、IL1b、IL6、NFkB和TNFa(用于标记炎症)和 I型和III型胶原蛋白及其m RNA、α-平滑肌肌动蛋白、转化生长因子β、脂氧合酶及其活性、羟脯氨酸(标记纤维化)。 除了基因操作外，我们还使用了新型小分子抑制剂C9-C9来抑制 Larp6与I型胶原基因5‘端SL单元的相互作用。相同的心功能指标 (超声心动图和有创插管产生PV曲线)用于野生型小鼠和终点 对心脏成纤维细胞进行炎症和纤维化标志物的检测。 这些数据将确定Larp6在酒精性心肌病中的作用，这是心脏成纤维细胞的特征 对酒精的反应，并说明Larp6如何影响炎症、纤维化、功能和结构性心脏 酒精性心肌病的研究进展。最终，这些研究可能确定新的治疗靶点 酒精性心肌病的治疗。