Identification of therapeutic target miRNAs involved in altered calcium handling in familial dilated cardiomyopathy

家族性扩张型心肌病钙处理改变中涉及的治疗靶标 miRNA 的鉴定

• 批准号: 9052020
• 负责人: Christine Wahlquist
• 金额: $ 5.43万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-03 至 2019-06-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9052020
• 关键词: ATP2A2; Address; Animals; Award; Biological Assay; Biological Process; Ca(2+)-Transporting ATPase; Calcium; Cardiac; Cardiac Myocytes; Cell Size; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Congenital cardiomyopathy; Contractile Proteins; Country; Diagnosis; Dilated Cardiomyopathy; Disease Progression; Disease susceptibility; Dose; Echocardiography; Equilibrium; Family member; Fibrosis; Functional disorder; Gene Targeting; Gene Transfer; Genes; Grant; Heart; Heart Diseases; Heart failure; Histology; Human; Image; Immunoprecipitation; In Vitro; Individual; Inherited; Kinetics; Learning; Lipids; Measures; MicroRNAs; Modeling; Molecular Profiling; Molecular Target; Monitor; Morbidity - disease rate; Mus; Mutation; Myocardial Ischemia; Myocardium; Pathway interactions; Patients; Plasmids; Proteins; RNA; Reporter; Research; Role; SERCA2a; Sarcomeres; Sarcoplasmic Reticulum; Signal Transduction; Testing; Therapeutic; Time; TimeLine; Tissues; Training; Transfection; Transgenic Model; Troponin T; Ventricular; Vesicle; Western Blotting; Work; base; deep sequencing; disease-causing mutation; early onset; expectation; familial dilated cardiomyopathy; functional restoration; gain of function; hemodynamics; imaging platform; improved; in vivo; induced pluripotent stem cell; inhibitor/antagonist; insight; intravenous injection; knock-down; loss of function; meetings; miRNA expression profiling; morphometry; mortality; mouse model; new therapeutic target; novel therapeutics; overexpression; public health relevance; research study; response; restoration; screening; small molecule; therapeutic development; therapeutic target; therapy development; uptake

 DESCRIPTION (provided by applicant): Heart disease remains the leading cause of morbidity and mortality in Western countries. While much is understood regarding development and treatment of ischemic heart disease, congenital cardiomyopathies are less understood. Dilated cardiomyopathy (DCM) is a form of heart disease that is characterized by ventricular dilation and systolic dysfunction. About 25-35% of DCM diagnoses are the result of inherited mutations in contractile proteins such as cardiac troponin T (cTn-T), which often culminate in early onset heart. Deficient calcium uptake due to decreased expression and activity of SERCA2a is a well-described hallmark of heart failure, and restoration by gene transfer has been shown to be effective in restoring contractility to human DCM iPSC derived cardiomyocytes (iPSC-CMs) to levels similar to that observed in control iPSC-CMs. Modeling DCM in patient hiPSCs offers an unprecedented opportunity to define the connection between the disease- causing mutation and the balance between adaptive and maladaptive signaling that dictates disease susceptibility and progression that at present are poorly understood. A high-throughput unbiased screen against a SERCA2a-eGFP reporter plasmid resulted in 144 microRNA (miR) hits that downregulated eGFP expression by >30%. These miRs were validated in a 5-point dose response assay and 82 were confirmed for their activity against SERCA2a. I have also profiled the expression of over 1000 miRs in human congenital DCM iPSC-CMs as compared to control iPSC-CMs from non-DCM family members. miRs found to be upregulated in DCM patient CMs were cross-referenced with those that target SERCA2a. The 23 miRs that met these criteria are now being tested for their effect against endogenous SERCA2a in control iPSC-CMs by calcium transient kinetic analysis and western blot. miRNA inhibition using anti-sense antagomiRs is also being performed in DCM hiPSC-CMs to improve endogenous SERCA2a and contractile function. miRs that show the most impressive results will be prioritized for in vivo inhibition in a mouse model of DCM. To test in vivo whether blocking pathologically upregulated miRNAs has a beneficial effect on SERCA2a expression and cardiac contractility, DCM mice will be administered a cocktail of the most potent angatomiRs, as judged by in vitro readouts. Mice will be monitored by echocardiogram every week for 3 months at which point hemodynamic analyses will be performed to determine PV loops and ESPVR. SERCA2a protein levels will be determined via western blot of cardiac tissue and histological sections will be analyzed for quantification of fibrosis and cells size relative to scramble sequence control antagomiR. The expectation is that DCM mice receiving a specific antagomiR cocktail will show enhanced cardiac function as compared to DCM mice receiving an inert control, and that the antagomiR cocktail may constitute a novel therapeutic option for the treatment of DCM.

 描述（由申请人提供）：心脏病仍然是西方国家发病率和死亡率的主要原因。虽然对缺血性心脏病的发展和治疗了解很多，但对先天性心肌病的了解较少。扩张型心肌病（DCM）是一种以心室扩张和收缩功能障碍为特征的心脏病。大约25-35%的DCM诊断是收缩蛋白如心肌肌钙蛋白T（cTn-T）的遗传突变的结果，其通常在早发性心脏中达到顶峰。由于SERCA 2a的表达和活性降低而导致的钙摄取不足是心力衰竭的充分描述的标志，并且已经显示通过基因转移的恢复有效地将人DCM iPSC衍生的心肌细胞（iPSC-CM）的收缩性恢复至与对照iPSC-CM中观察到的水平相似的水平。在患者hiPSC中建模DCM提供了前所未有的机会来定义致病突变与适应性和适应不良信号传导之间的平衡之间的联系，适应性和适应不良信号传导决定了目前尚不清楚的疾病易感性和进展。针对SERCA 2a-eGFP报告质粒的高通量无偏筛选导致144个microRNA（miR）命中，其下调eGFP表达> 30%。这些miR在5点剂量反应测定中得到验证，并且82个miR被确认具有针对SERCA 2a的活性。我还分析了与来自非DCM家族成员的对照iPSC-CM相比，人先天性DCM iPSC-CM中超过1000个miR的表达。发现在DCM患者CM中上调的miR与靶向SERCA 2a的miR交叉参考。现在通过钙瞬时动力学分析和蛋白质印迹测试符合这些标准的23种miR在对照iPSC-CM中对内源性SERCA 2a的作用。还在DCM hiPSC-CM中进行使用反义miR的miRNA抑制以改善内源性SERCA 2a和收缩功能。显示最令人印象深刻的结果的miR将优先用于DCM小鼠模型中的体内抑制。为了在体内测试阻断病理上上调的miRNA是否对SERCA 2a表达和心脏收缩性具有有益作用，将向DCM小鼠施用最有效的angatomiR的混合物，如通过体外读数判断的。每周通过超声心动图监测小鼠，持续3个月，此时将进行血液动力学分析以确定PV环和ESPVR。将通过心脏组织的蛋白质印迹测定SERCA 2a蛋白水平，并将分析组织学切片以定量相对于乱序序列对照的纤维化和细胞大小。预期与接受惰性对照的DCM小鼠相比，接受特定antagomiR混合物的DCM小鼠将显示出增强的心脏功能，并且antagomiR混合物可能构成治疗DCM的新型治疗选择。