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

致心律失常性心肌病是一种心肌遗传性疾病， 是室性心律失常、猝死、心肌纤维肥胖症、细胞死亡和心力衰竭。ACM是由 主要是通过编码桥粒蛋白（包括桥粒斑蛋白（DSP））的基因突变。在心中， 桥粒主要存在于肌细胞中，并负责心肌的机械完整性。他们是 也是机械传感和转导的枢纽，包括Hippo和经典WNT（cWNT）途径， 它们与ACM有关。桥粒蛋白质也在心外膜中表达，心外膜是心脏的起始部位。 ACM表型的表现。心外膜细胞在ACM发病机制中的具体作用尚不清楚。 cWNT通路已成为ACM中有吸引力的治疗靶点。实验数据， 然而，是相互冲突的，范围从抑制到激活的cWNT在ACM的频谱。的 沿着这种主要调节途径的系统靶向的潜在偶然效应的模糊性已经引起了 相当大的恐慌，导致ACM中cWNT途径的治疗靶向陷入僵局。 我们提供了ACM在人类和小鼠心脏中的数据，表明这种模糊性部分是由于 测定细胞异质性心肌中的cWNT活性。因此，尽管cWNT是 在心肌细胞中激活，整个心肌暴露于增加水平的分泌型cWNT抑制剂 （cWNT-Is）。在具有ACM的人类心脏中，26个cWNT-Is的转录水平的失调是显著的 增加，包括SFRP 3蛋白，一种经典的cWNT-I。为了解决不确定性，我们将使用 特异性激活或抑制心肌细胞中cWNT通路的非定向方法， 心外膜细胞并确定表型效应，包括对分泌的cWNT-Is表达的效应。 诱导型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 同时使用功能丧失和获得的b-连环蛋白小鼠抑制或激活cWNT。心脏 功能、心律失常、细胞凋亡、纤维脂肪形成和基因表达，包括肌细胞中的分泌蛋白组， 将表征心外膜细胞。将使用双报告基因对DSP+/-心外膜细胞进行条件性标记 （Rosa 26 mT/mG）小鼠及其向其他心脏细胞的分化将通过命运作图进行分析。标记的细胞 将分离衍生自Dsp+/-心外膜细胞的细胞，并通过单细胞RNA-Seq分析以确定它们的 转录同一性并鉴定由心外膜衍生细胞的每个子集表达的分泌组。同样地， 激活或抑制cWNT途径对Dsp+/-心外膜细胞分化为其他细胞的影响 心脏细胞类型和分泌组将使用功能获得和丧失的b-连环蛋白小鼠来确定。 这些发现将确定cWNT的细胞类型特异性靶向是否是有益的治疗方法 ACM的方法，将定义心外膜细胞在ACM中的作用，并可能导致识别分泌的 可能作为ACM诊断和预后生物标志物和治疗靶点的因素。

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.