Molecular and cellular functions of Ano5 in heart

描述（由申请方提供）：质膜完整性对细胞稳态和功能至关重要。质膜的物理、化学或代谢破坏导致细胞中的修复或死亡紧急情况。因此，有效的质膜修复机制对于生命是必不可少的，因为由于基因突变导致的该过程的破坏可导致许多疾病，包括肌营养不良症和相关的心肌病。我们和其他人的研究表明，心肌细胞的膜修复反应是由包括dysferlin和MG 53在内的几种蛋白介导的。然而，这一重要生理过程的分子机制尚未完全确定。我们的初步数据发现，anoctamin 5（Ano 5）在肌细胞膜修复中起着重要作用。Ano 5属于anoctamin蛋白家族，该家族包括至少十种蛋白质，它们都具有八个跨膜结构域，具有已证实或推定的钙激活氯离子通道（CaCC）功能。ANO 5基因（编码ANO 5）的突变导致人类患者的肌营养不良症。然而，目前对Ano 5在心肌细胞中的分子和细胞功能知之甚少，并且对Ano 5介导的膜修复的分子机制仍然知之甚少。这项研究计划的长期目标是了解Ano 5在心脏生理和疾病中的分子和细胞机制。在初步研究中，我们发现Ano 5主要位于内质网/肌浆网（ER/SR）上，并且Ano 5的RNAi沉默显示肌细胞中的膜修复缺陷。因此，我们的数据为Ano 5在肌肉细胞的细胞生理学中提供了一种新的生物学功能。在这个项目中，我们将专注于验证Ano 5参与钙激活氯离子通道（CaCC）活性并在心肌细胞质膜修复中发挥重要作用的假设。通过调控Ano 5的表达，以及使用活细胞成像、生化标记物、离体和体内动物模型研究，我们计划的实验将显著推进对心肌细胞膜修复潜在机制的理解，并开始定义用于调节膜修复能力以治疗与异常膜稳定性相关的疾病的潜在治疗靶点。质膜完整性的破坏是许多疾病的基础，包括心肌病。我们的项目旨在了解Ano 5在肌肉生理学和疾病中的分子和细胞功能。这些研究将有助于通过调节Ano 5介导的膜修复能力来确定治疗与质膜完整性受损相关的心脏病的治疗靶点。

DESCRIPTION (provided by applicant): The plasma membrane integrity is of critical importance for cell homeostasis and function. Physical, chemical or metabolic disruption of the plasma membrane leads to a repair-or-die emergency in the cell. Thus, an efficient plasma membrane repair mechanism is essential for life since disruption of this process due to genetic mutations can result in a number of diseases including muscular dystrophy and associated cardiomyopathy. Previous studies from others and us demonstrated that the membrane repair response in cardiomyocytes is mediated by several proteins including dysferlin and MG53. However, the molecular mechanisms underlying this important physiological process have not been fully defined. Our preliminary data found that anoctamin 5 (Ano5) plays an essential role in membrane repair in myocytes. Ano5 belongs to the anoctamin protein family that includes at least ten proteins all possessing eight transmembrane domains with proved or putative calcium-activated chloride channel (CaCC) functions. Mutations in the ANO5 gene (encoding Ano5) lead to muscular dystrophies in human patients. However, there is little known about the molecular and cellular functions of Ano5 in cardiomyocytes and the molecular mechanisms underlying Ano5-mediated membrane repair remain poorly understood. The long-term goal of this research proposal is to understand the molecular and cellular mechanisms for Ano5 in heart physiology and disease. In pilot studies, we found that Ano5 is primarily localized on the endoplasmic/sarcoplasmic reticulum (ER/SR) and RNAi-silencing of Ano5 shows defective membrane repair in myocytes. Thus, our data present a new biological function for Ano5 in the cellular physiology of muscle cells. In this project, we will focus on testing the hypothesis that Ano5 is involved in the calcium-activated chloride channel (CaCC) activity and plays an essential role in plasma membrane repair of cardiomyocytes. Through manipulating expression of Ano5 and the use of live cell imaging, biochemical markers, ex vivo and in vivo animal model studies, our planned experiments will significantly advance understanding of the mechanisms underlying membrane repair of cardiomyocytes, and begin to define potential therapeutic targets for the regulation of membrane repair capacity to treat the diseases associated with abnormal membrane stability. Disrupted plasma membrane integrity underlies a number of diseases including cardiomyopathy. Our project is designed to understand the molecular and cellular functions of Ano5 in muscle physiology and disease. These studies will aid in defining therapeutic target for the treatment of treatment of heart diseases associated with compromised plasma membrane integrity through the regulation of Ano5-mediated membrane repair capacity.