Ca2+ and ROS Crosstalk Signaling in Cardiac Mitochondria

描述(申请人提供)：这项建议的长期目标是建立一个统一的理论来描述心肌细胞中钙离子和活性氧物种(ROS)之间的串扰信号机制，并将这些信号机制转化为心功能的生理学和病理学。线粒体Ca~(2+)和ROS在调节心肌细胞生死中的关键作用已得到公认。线粒体介导的生命和死亡之间的分离存在于钙离子和ROS浓度之间的最佳平衡。该领域现有的研究大多集中在钙离子或ROS动态平衡上。然而，这两条信号通路之间的相互作用才刚刚开始受到少数实验室的关注。有趣的是，越来越多的文献表明线粒体动力学(分裂、融合和运输)在细胞ATP、Ca~(2+)和ROS稳态的生理调节中发挥着重要作用。在这个方案中，我们将研究这三个重要组成部分(钙离子、ROS和线粒体分裂机制)是如何通过通讯来调节心脏钙离子和ROS串扰信号的。我们将综合运用细胞生物学(如共聚焦显微镜)、分子生物学(如基因转移)、生物化学(如Western blots)和转基因小鼠模型(如亲环素D(CypD)基因敲除小鼠和mt-cpYFP转基因小鼠)等多学科方法来阐明ROS和钙离子共生的机制，重点是线粒体裂变蛋白DLP1和线粒体通透性转变(MPT)。我们的中心假设是：线粒体钙离子浓度([Ca2+]m)的增加有利于线粒体分裂动力学的平衡，进而增加ROS的产生。由此产生的氧化环境导致额外的线粒体钙离子内流。[Ca~(2+)]_m和ROS的增加都增加了MPT的开放概率，从而进一步增加了ROS的产生。最终，这种正反馈环路被钙离子和ROS激活的线粒体钙外流机制(包括Na/Ca交换和MPT)所抵消。这三个具体目标是：1)确定增加的[Ca~(2+)]m是否促进线粒体的分裂过程，从而导致ROS的产生增加。2)探讨含MPT途径的CypD在[Ca~(2+)]_m介导的ROS生成中的作用。3)探讨MPT作为线粒体钙离子快速外流机制的作用。 公共卫生相关性：氧化应激可导致多种人类疾病，包括慢性心力衰竭心肌病、缺血性心脏病、神经退行性疾病、糖尿病、肥胖和衰老。这项研究的重点是阐明细胞内产生活性氧物种的机制。我们的目标是，不仅对氧化应激介导的疾病机制做出科学贡献，而且开发可能的治疗方法来治疗这些衰弱的疾病。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to establish a unified theory to describe the mechanisms of crosstalk signaling between Ca2+ and reactive oxygen species (ROS) in cardiac muscle cells, and to translate these signaling mechanisms to the physiology and pathology of cardiac function. The pivotal role of mitochondrial Ca2+ and ROS in mediating the life and death of cardiac muscle cells is well recognized. The separation between mitochondria-mediated life versus death resides in the finest balance between concentrations of Ca2+ and ROS. The majority of existing research in the field focuses individually either on Ca2+ or ROS homeostasis. The interaction between these two signaling pathways, however, has just begun to gain attention by a small number of laboratories. Intriguingly, there is a growing library of literature suggesting that mitochondrial dynamics (fission, fusion, and trafficking) play an essential role in the physiological regulation of cellular ATP, Ca2+, and ROS homeostasis. In this proposal, we will study how these three important components (Ca2+, ROS, and mitochondrial fission machinery) communicate to regulate cardiac Ca2+ and ROS crosstalk signaling. We will use a multidisciplinary approach encompassing techniques of cell biology (e.g. confocal microscopy), molecular biology (e.g. gene transfer), biochemistry (e.g. western blots), and transgenic mouse models (e.g. cyclophilin D (CypD) knockout mice and mt-cpYFP transgenic mice) to elucidate the mechanisms of ROS and Ca2+ symbiosis with an unique emphasis on mitochondrial fission protein DLP1 and mitochondrial permeability transition (MPT). Our central hypothesis is: an increased mitochondrial Ca2+ concentration ([Ca2+]m) favors the balance of mitochondrial dynamics towards fission that in turn increases ROS generation. The resulting oxidized environment leads to additional mitochondrial Ca2+ influx. Both the increases in [Ca2+]m and ROS enhance the opening probability of MPT that further augments ROS generation. Eventually, this positive feedback loop is counter balanced by Ca2+ and ROS activated mitochondrial Ca2+ efflux mechanisms including Na/Ca exchange and MPT. The three specific aims are: 1) To determine whether an increased [Ca2+]m promotes mitochondrial fission processes, which then lead to increase ROS generation. 2) To assess the contribution of a CypD containing MPT pathway in [Ca2+]m-mediated ROS generation. 3) To determine the role of MPT as a rapid Ca2+ efflux mechanism of mitochondria. PUBLIC HEALTH RELEVANCE: Oxidative stress can cause numerous human diseases including cardiomyopathy in chronic heart failure, ischemic heart disease, neurodegenerative diseases, diabetes, obesity, and aging. The proposed research focuses on the elucidation of cellular mechanisms of reactive oxygen species generation. It is our objective, not only to make a scientific contribution to oxidative stress-mediated disease mechanisms, but also to develop possible therapeutic means for treating these debilitating disorders.