Cell type-Specific Therapeutic Targeting of canonical WNT Pathway in Arrhythmogenic Cardiomyopathy

致心律失常性心肌病典型 WNT 通路的细胞类型特异性治疗靶向

• 批准号: 10418626
• 负责人: Ali J Marian
• 金额: $ 49.36万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-10 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10418626
• 关键词: Apoptosis; Arrhythmia; Arrhythmogenic Right Ventricular Dysplasia; Biological; Biological Assay; Cardiac; Cardiac Myocytes; Cardiomyopathies; Caspase; Cell Death; Cells; Conflict (Psychology); Connexin 43; Data; Desmosomes; Development; Diagnosis; Disease; Echocardiography; Electrophysiology (science); Epicardium; Epithelial; Exposure to; Gene Expression; Genes; Genetic; Genetic Diseases; Genetic Transcription; Heart; Heart failure; Histologic; Human; In Situ Nick-End Labeling; Intercalated disc; Intervention; Knowledge; Lead; Malignant Neoplasms; Mechanics; Molecular; Monitor; Mus; Muscle Cells; Mutation; Myocardial; Myocardium; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Phenotype; Prognostic Marker; Proteins; Publishing; Regulatory Pathway; Reporter; Reporting; Risk; Role; Signal Transduction; Site; Sodium Channel; Sudden Death; Tamoxifen; Techniques; Therapeutic; Transcript; Uncertainty; Ventricular; Ventricular Arrhythmia; arrhythmogenic cardiomyopathy; beta catenin; causal variant; cell type; circulating biomarkers; desmoplakin; diagnostic biomarker; effective therapy; frizzled related protein-3; gain of function; heart dimension/size; heart function; inhibitor; lipid biosynthesis; loss of function; mechanotransduction; mouse model; novel diagnostics; paracrine; prognostic; single cell analysis; single-cell RNA sequencing; therapeutic target; transcriptome sequencing

Arrhythmogenic cardiomyopathy (ACM) is a myocardial genetic disease whose cardinal manifestations are ventricular arrhythmias, sudden death, myocardial fibro-adiposis, cell death, and heart failure. ACM is caused primarily by mutations in genes encoding desmosome proteins, including desmoplakin (DSP). In the heart, desmosomes are mainly present in myocytes and are responsible for myocardial mechanical integrity. They are also hubs for mechanosensing and transduction, including the Hippo and canonical WNT (cWNT) pathways, which are involved in ACM. Desmosome proteins are also expressed in the epicardium, the site of initial manifestations of ACM phenotype. Specific role(s) of the epicardial cells in the pathogenesis of ACM is unknown. The cWNT pathway has emerged as an attractive therapeutic target in ACM. Experimental data, however, are conflicting, with a spectrum ranging from suppression to activation of the cWNT in ACM. The ambiguity along with potential fortuitous effects of systemic targeting of this major regulatory pathway have raised considerable trepidations, leading to an impasse on therapeutic targeting of the cWNT pathway in ACM. We provide data in human and mouse hearts with ACM suggesting that the ambiguity is in part due to determining the cWNT activity in cellularly heterogeneous myocardium. Accordingly, whereas the cWNT is activated in cardiac myocytes, the whole myocardium is exposed to increased levels of secreted cWNT inhibitors (cWNT-Is). The dysregulation is remarkable in the human hearts with ACM as transcript levels of 26 cWNT-Is are increased, including SFRP3 protein, a classic cWNT-I. To resolve the uncertainty, we will use an uncommitted approach of activating or suppressing the cWNT pathway specifically in cardiac myocytes and epicardial cells and determining the phenotypic effects, including effects on expression of secreted cWNT-Is. Inducible Cre-deleter mice will be used to delete Dsp in cardiac myocytes or epicardial cells post-natally while concomitantly suppressing or activating the cWNT using loss- and gain-of-function b-catenin mice. Cardiac function, arrhythmias, apoptosis, fibro-adipogenesis and gene expression, including secretome in myocytes and epicardial cells will be characterized. Dsp+/- epicardial cells will be conditionally tagged using the dual reporter (Rosa26mT/mG) mice and their differentiation to other cardiac cells will be analyzed by fate mapping. Tagged cells derived from Dsp+/- epicardial cells will be isolated and analyzed by single cell RNA-Seq to determine their transcriptional identity and identify secretome expressed by each subset of epicardial-derived cells. Likewise, effects of activation or suppression of the cWNT pathway on differentiation of the Dsp+/- epicardial cells to other cardiac cell types and the secretome will be determined using gain- and loss-of-function b-catenin mice. The findings will determine whether cell-type specific targeting of the cWNT is a beneficial therapeutic approach in ACM, will define the role of epicardial cells in ACM, and could lead to identification of secreted factors that might serve as diagnostic and prognostic biomarkers and therapeutic targets in ACM.

心律失常心肌病（ACM）是一种心肌遗传疾病，其基本表现 是心室心律失常，猝死，心肌纤维腺发素病，细胞死亡和心力衰竭。 ACM是引起的 主要是通过编码脱骨蛋白的基因突变，包括去毛蛋白（DSP）。在心中 脱染体主要存在于心肌细胞中，并负责心肌机械完整性。他们是 还用于机械连感和转导的枢纽，包括河马和规范Wnt（CWNT）途径， 参与ACM。脱骨蛋白也在心外膜中表达，初始位置 ACM表型的表现。心外膜细胞在ACM发病机理中的特定作用尚不清楚。 CWNT途径已成为ACM中有吸引力的治疗靶标。实验数据， 然而，矛盾的是，频谱从抑制到ACM中CWNT的激活。这 歧义以及这一主要调节途径的全身靶向的潜在偶然效应已经提高 相当大的恐惧，导致对ACM中CWNT途径的治疗靶向僵局。 我们以ACM提供人类和小鼠心脏的数据，表明歧义是部分原因是 确定细胞异质性心肌的CWNT活性。因此，cwnt是 在心肌细胞中激活，整个心肌暴露于分泌的CWNT抑制剂的水平升高 （CWNT-IS）。在ACM的人心中，功能障碍是26 cwnt-is的转录水平 增加了，包括经典的CWNT-I，包括SFRP3蛋白。为了解决不确定性，我们将使用 未能在心肌细胞中专门激活或抑制CWNT途径的方法 心外膜细胞并确定表型作用，包括对分泌CWNT-IS表达的影响。 可诱导的Cre-Deleter小鼠将在后产后删除心肌细胞或心外膜细胞中的DSP 同时使用功能损失和功能获得的B-catenin小鼠同时抑制或激活CWNT。心脏 功能，心律不齐，凋亡，纤维辅助生成和基因表达，包括肌细胞中的分泌组和 心外膜细胞将被表征。 DSP +/-心外膜细胞将使用双重报告程序进行条件标记 （ROSA26MT/MG）小鼠及其与其他心脏细胞的分化将通过命运图分析。标记的单元格 源自DSP +/-心外膜细胞将通过单细胞RNA-Seq分离并分析，以确定其 转录身份并确定由心外膜衍生细胞的每个子集表示的分泌组。同样地， 激活或抑制CWNT途径对DSP +/-心外膜细胞分化为其他的影响 心脏细胞类型和分泌组将使用功能和功能丧失B-catenin小鼠确定。 这些发现将确定CWNT的细胞类型特异性靶向是有益的治疗方法 ACM中的方法将定义心外膜细胞在ACM中的作用，并可能导致分泌的鉴定 ACM中可能用作诊断和预后生物标志物和治疗靶标的因素。