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

 描述(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有很多了解，但对先天性心肌病了解较少。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诊断是由心肌肌钙蛋白T(cTn-T)等收缩蛋白的遗传突变造成的，这种突变通常会导致早发心脏。由于SERCA2a的表达和活性降低导致的钙摄取不足是心力衰竭的一个众所周知的标志，通过基因转移恢复人DCM iPSC来源的心肌细胞(iPSC-CMS)的收缩能力已被证明有效地恢复到与对照IPSC-CMS相似的水平。在患者的HiPSCs中建立DCM模型提供了一个前所未有的机会来定义致病突变和适应性和非适应性信号之间的平衡之间的联系，适应性和非适应性信号决定了疾病的易感性和进展，目前人们对此知之甚少。对SERCA2a-EGFP报告质粒进行高通量无偏筛选，得到144个microRNA(MiR)，下调了30%的EGFP表达。这些MIR在5点剂量反应试验中得到验证，82个MIR被证实具有抗SERCA2a的活性。我还分析了1000多个miRs在人类先天性DCM IPSC-CMS中的表达，与来自非DCM家族成员的对照IPSC-CMS相比。在DCM患者CMS中发现的MIR上调与以SERCA2a为靶点的MIR进行了交叉参考。满足这些标准的23个miRs现在正在通过钙瞬变动力学分析和蛋白质印迹来测试它们对内源性SERCA2a在控制IPSC-CMS中的作用。在DCM hiPSC-CMS中，也正在使用反义抗体素抑制miRNA，以改善内源性SERCA2a和收缩功能。显示最令人印象深刻的结果的MIR将在DCM的小鼠模型中优先用于体内抑制。为了在体内测试阻断病理上调的miRNAs是否对SERCA2a表达和心肌收缩能力有好处，根据体外读数判断，将给DCM小鼠注射最有效的血管紧张素受体的鸡尾酒。每周对小鼠进行超声心动图监测，持续3个月，然后进行血流动力学分析，以确定PV环和ESPVR。SERCA2a蛋白水平将通过心脏组织的蛋白质印迹来确定，组织切片将被分析以量化纤维化和与扰乱序列控制的AnagomiR相关的细胞大小。人们的期望是，与接受惰性对照的DCM小鼠相比，接受特定antagomiR鸡尾酒的DCM小鼠将显示出增强的心功能，并且antagomiR鸡尾酒可能构成治疗DCM的一种新的治疗选择。

 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.