Molecular and cellular functions of Ano5 in heart

描述(由申请人提供)：质膜的完整性对于细胞的稳态和功能至关重要。质膜的物理、化学或新陈代谢的破坏会导致细胞的“要么修复要么死亡”的紧急情况。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中断可能会导致许多疾病，包括肌营养不良症和相关的心肌病。我们和其他人之前进行的研究表明，几种蛋白质，包括deferlin和MG53，介导了心肌细胞的膜修复反应。然而，这一重要生理过程背后的分子机制还没有完全确定。我们的初步数据发现，Anoctamin 5(ANO5)在心肌细胞的膜修复中起着至关重要的作用。ANO5属于Anoctamin蛋白家族，包括至少10个蛋白，每个蛋白都有8个跨膜结构域，具有已证实或可能具有钙激活氯通道(CACC)功能。ANO5基因(编码ANO5)的突变会导致人类患者的肌肉营养不良。然而，目前对Ano5在心肌细胞中的分子和细胞功能知之甚少，Ano5介导的膜修复的分子机制也知之甚少。这项研究计划的长期目标是了解ANO5在心脏生理学和疾病中的分子和细胞机制。在前期研究中，我们发现Ano5主要定位于内质/肌浆网(ER/SR)，而对Ano5的RNAi沉默表明心肌细胞膜修复存在缺陷。这表明Ano5在肌肉细胞的细胞生理中具有新的生物学功能。在这个项目中，我们将重点检验这样的假设，即Ano5通过肌浆网(SR)上的寡聚参与钙激活的氯通道(CACC)的活动，以及Ano5通过促进膜损伤时产生小泡而在心肌细胞的质膜修复中发挥重要作用。我们计划的实验将通过操纵Ano5的表达和使用活细胞成像、生化标记物、体外和体内动物模型研究来显著促进对心肌细胞膜修复机制的理解。这些数据将开始确定膜修复调节的潜在治疗靶点，从而治疗与膜稳定性异常相关的疾病。质膜完整性受损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在肌肉生理学和疾病中的分子和细胞功能。这些研究将有助于确定通过调节ANO5介导膜修复能力来治疗与质膜完整性受损相关的心脏病的治疗目标。

DESCRIPTION (provided by applicant): 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. An efficient plasma membrane repair mechanism is essential for life because disruption of this process due to genetic mutations can result in a number of diseases including muscular dystrophy and associated cardiomyopathy. Previous studies conducted by ourselves and others demonstrate that several proteins, including dysferlin and MG53, mediate the membrane repair response in cardiomyocytes. However, the molecular mechanisms underlying this important physiological process has 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. This shows a new biological function of 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 through oligomerization on the sarcoplasmic reticulum (SR), and that Ano5 plays an essential role in plasma membrane repair of cardiomyocytes by promoting vesicle generation upon membrane damage. Our planned experiments will significantly advance understanding of cardiomyocyte membrane repair mechanisms by manipulating expression of Ano5 and the use of live cell imaging, biochemical markers, ex vivo and in vivo animal model studies. These data will begin to define potential therapeutic targets for the regulation of membrane repair, thereby treating 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 targets for the treatment of heart diseases associated with compromised plasma membrane integrity through the regulation of Ano5-mediated membrane repair capacity.