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.

致心律失常型心肌病(ACM)是一种以心脏遗传性疾病为主要表现的疾病 室性心律失常、猝死、心肌纤维化、细胞死亡和心力衰竭。ACM是由 主要是通过编码桥粒蛋白的基因突变，包括桥粒蛋白(DSP)。在内心深处， 桥粒主要存在于心肌细胞中，与心肌的机械完整性有关。他们是 也是机械传感和转导的中枢，包括河马和典型的WNT(CWNT)通路， 它们都参与了ACM。桥粒蛋白也在心外膜中表达，心外膜是最初的 ACM表型的表现。心外膜细胞在急性心肌梗死发病机制中的具体作用(S)尚不清楚。 CWNT通路已成为ACM治疗的重要靶点。实验数据， 然而，它们是相互冲突的，在ACM中的光谱范围从抑制到激活cWNT。这个 对这一主要调控途径的系统性靶向的模棱两可以及潜在的偶然影响，已经引起了人们的关注 相当多的颤抖，导致在ACM中cWNT通路的治疗靶点方面陷入僵局。 我们用ACM提供了人类和小鼠心脏的数据，表明这种模棱两可的部分原因是 细胞异质性心肌中cWNT活性的测定。因此，鉴于cwnt是 在心肌细胞中被激活，整个心肌暴露在分泌水平增加的cWNT抑制物中 (CWNT-IS)。这种失调在人的心脏中是显著的，ACM作为26个cWNT-IS的转录水平 增加的包括sFRP3蛋白，一种经典的cWNT-I。为了解决不确定性，我们将使用 在心肌细胞中激活或抑制cWNT通路的未确定的方法 并测定表型效应，包括对分泌的cWNT-IS表达的影响。 可诱导的Cre-deleter小鼠将用于在出生后删除心肌细胞或心外膜细胞中的DSP 同时同时使用功能丧失和获得的b-连环蛋白抑制或激活cWNT。心脏 功能，心律失常，细胞凋亡，纤维脂肪形成和基因表达，包括心肌细胞和 心外膜细胞的特征将被描述。将使用双重报告程序有条件地标记DSP+/-心外膜细胞 (Rosa26mT/mg)小鼠及其向其他心肌细胞的分化将通过命运图谱进行分析。标记的单元格 将分离来自于DSP+/-心外膜细胞，并用单细胞RNA-Seq分析其 转录鉴定和鉴定心外膜来源细胞各亚群表达的分泌组。同样， 激活或抑制cWNT通路对DSP+/-心外膜细胞分化的影响 心肌细胞类型和分泌体将使用功能增强和丧失功能的b-catenin小鼠来确定。 这些发现将确定针对cWNT的细胞类型特异性靶向是否有益于治疗 在ACM中，将明确心外膜细胞在ACM中的作用，并可能导致对分泌的 可作为ACM的诊断和预后生物标志物和治疗靶点的因素。