Stem cells and the fibroblast/adipocyte lineage in arrhythmogenic cardiomyopathy

描述（由申请人提供）：本拨款申请是对主题15-OD (ORDR)-101的回应。心律失常性右室心肌病（ARVC）是一种遗传性心脏病，其特征是严重的室性心律失常，以及纤维和脂肪组织逐渐取代健康心肌，在许多情况下导致严重心力衰竭，在没有心脏移植的情况下导致死亡。ARVC与桥粒体蛋白的突变有关，最显著的是在plakophilin-2 （PKP2）的基因编码中。在桥粒断裂后，心肌细胞发生的变化引起了广泛的关注。然而，基于肌细胞的实验模型未能重现该疾病的纤维脂肪浸润特征。在这里，我们从以心肌细胞为中心的假设转移到提出一个新的范式：纤维脂肪浸润起源于非心肌细胞的行为。我们的研究重点集中在两种能够产生心脏成纤维细胞谱系的干细胞群体：包括心外膜的上皮细胞和造血干细胞。通过使用新的小鼠分子遗传策略和细胞生物学分析，我们将验证这样的假设，即这些干细胞中桥粒体完整性的丧失会导致心脏中成纤维细胞和脂肪细胞数量的增加，而代价是心肌质量的减少。具体目标是：1.；探讨PKP2在心外膜源性细胞迁移、增殖和分化中的作用。2. 建立心外膜特异性条件表达arvc相关突变的小鼠模型。3. 生成并表征造血干细胞特异性PKP2缺陷的动物模型。这些研究为更好地理解病变心脏成纤维细胞生物学提供了新的假设和新的实验模型。此外，我们的实验开启了一种诱人的可能性，即在未来，这些干细胞群（造血细胞；心外膜细胞）可以被靶向治疗，以降低心肌质量损失的速度，防止ARVC患者心力衰竭的发生。此外，尽管ARVC确实是一种“罕见疾病”，但心脏纤维化是一种常见的重要医学疾病，通过操纵成纤维祖细胞来控制纤维化在很大程度上仍未被探索。在本赠款下产生的实验模型可以有助于缩小这一特定的知识差距。干细胞后代可追溯的动物模型将成为整个科学界的可用工具。这些动物将被用于其他实验，在这些实验中，获得性疾病（例如，LAD闭塞；起搏诱导的肥大）被建模，从而可以确定所产生的成纤维细胞的来源。因此，这个项目与arvc患者的小社区直接相关。然而，正如对罕见疾病的研究经常出现的情况一样，从本项目中吸取的经验教训很可能会被更广泛的心脏病患者群体所应用，心脏病是美国的头号杀手。

DESCRIPTION (provided by applicant): This grant application is a response to Topic 15-OD (ORDR)-101. Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is an inherited cardiac disease characterized by severe ventricular arrhythmias, as well as progressive replacement of healthy myocardium with fibrous and fatty tissue leading in many cases to severe heart failure and, in the absence of a heart transplant, death. ARVC has been associated with mutations in desmosomal proteins, most prominently in the gene coding for plakophilin-2 (PKP2). Much attention has been given to the changes that occur in cardiac myocytes following disruption of the desmosome. Yet, myocyte-based experimental models have failed to reproduce the fibrofatty infiltration characteristic of the disease. Here, we shift away from cardiomyocyte-centered hypotheses to propose a new paradigm: that the fibrofatty infiltration has its origins in the behavior of non-myocyte cells. Our focus centers on two stem cell populations capable of generating a cardiac fibroblast lineage: the epithelial cells that comprise the epicardium, and the hematopoietic stem cells. Through the use of novel mouse molecular genetic strategies, and cell biological assays, we will test the hypothesis that loss of desmosomal integrity in these stem cells leads to an increase in the population of fibroblasts and adipocytes in the heart, at the expense of myocardial mass. Specific aims are: 1. To assess the role of PKP2 on migration, proliferation and differentiation of epicardium-derived cells. 2. To generate a murine model of epicardium-specific conditional expression of an ARVC-relevant mutation. 3. To generate and characterize an animal model of hematopoietic stem cell-specific PKP2 deficiency. These studies offer new hypotheses and new experimental models to better understand fibroblast biology in the diseased heart. Moreover, our experiments open the tantalizing possibility that in the future, these stem-cell populations (hematopoietic; epicardial) can be targeted for treatment, to decrease the rate of loss of myocardial mass and prevent the occurrence of heart failure in patients with ARVC. Moreover, although ARVC is, indeed, a "rare disease," cardiac fibrosis is a common occurrence of major medical importance, and control of fibrosis via manipulation of the fibroblast progenitor cells remains largely unexplored. Experimental models generated under the present grant can be instrumental to reduce that particular knowledge gap. Animal models where the stem cell progeny is traceable will become available to the scientific community as a whole. These animals will be utilized on additional experiments where acquired diseases (e.g., LAD occlusion; pacing-induced hypertrophy) are modeled, thus allowing identification of the origin of the resulting fibroblasts. As such, this project is directly relevant to the small community of ARVC-afflicted patients. Yet, as it is often the case with the study of rare diseases, lessons learned from the present project are likely to find applications relevant to the much broader community of patients afflicted with heart disease, the number one killer in the United States. PUBLIC HEALTH RELEVANCE: Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is a devastating, incurable disease of the heart that can run in families. Patients suffer very severe arrhythmias and are at great risk of sudden death at a young age. As the disease progresses, the heart muscle weakens and becomes replaced with fatty and fibroid tissue, to the point where the heart is unable to pump blood. In the terminal stages (often in young adults), heart transplant becomes the only alternative. Here, we propose that part of the origins of the disease may involve a particular type of stem cells, which may produce excess fatty and fibroid cells. To understand the role of these stem cells in ARVC, and potentially manipulate their function to preserve the heart wall, are the main goals of this grant.