Molecular and cellular functions of Ano5 in heart

描述（由申请人提供）：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出现“要么修复，要么死亡”的紧急情况。因此，有效的质膜修复机制对生命至关重要，因为由于基因突变导致的这一过程的破坏可能导致许多疾病，包括肌肉萎缩症和相关的心肌病。我们和其他人之前的研究表明，心肌细胞的膜修复反应是由几种蛋白质介导的，包括dysferlin和MG53。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Ano5在肌细胞的膜修复中起着至关重要的作用。Ano5属于氨基酸蛋白家族，该家族包括至少10个蛋白质，它们都具有8个跨膜结构域，具有已证实或推测的钙活化氯离子通道（CaCC）功能。ANO5基因（编码ANO5）的突变导致人类患者肌肉营养不良。然而，对Ano5在心肌细胞中的分子和细胞功能知之甚少，并且Ano5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Ano5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Ano5主要定位于内质/肌浆网（ER/SR），而Ano5的rnai沉默显示肌细胞的膜修复缺陷。因此，我们的数据提出了Ano5在肌肉细胞生理中的一种新的生物学功能。在本项目中，我们将重点验证Ano5参与钙活化氯离子通道（calcium-activated chloride channel, CaCC）活性并在心肌细胞质膜修复中发挥重要作用的假设。通过操纵Ano5的表达，利用活细胞成像、生化标记、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机制的理解，并开始确定调节膜修复能力的潜在治疗靶点，以治疗与异常膜稳定性相关的疾病。质膜完整性的破坏是包括心肌病在内的许多疾病的基础。我们的项目旨在了解Ano5在肌肉生理和疾病中的分子和细胞功能。这些研究将有助于通过调节ano5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.