Ca2+ and ROS Crosstalk Signaling in Cardiac Mitochondria

描述（由申请人提供）：本提案的长期目标是建立一个统一的理论来描述心肌细胞中Ca2+和活性氧（ROS）之间的串扰信号传导机制，并将这些信号传导机制转化为心功能的生理和病理。线粒体Ca2+和ROS在介导心肌细胞生死中的关键作用已得到充分认识。线粒体介导的生命与死亡之间的分离存在于Ca2+和ROS浓度之间的最佳平衡中。该领域的大多数现有研究都单独关注Ca2+或ROS稳态。然而，这两种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（裂变、融合和运输）在细胞ATP、Ca2+和ROS稳态的生理调节中起着重要作用。在本提案中，我们将研究这三种重要成分（Ca2+， ROS和线粒体裂变机制）如何沟通以调节心脏Ca2+和ROS串扰信号。我们将使用多学科方法，包括细胞生物学（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（如亲环蛋白D （CypD）敲除小鼠和mt-cpYFP转基因小鼠）来阐明ROS和Ca2+共生的机制，并特别强调线粒体分裂蛋白DLP1和线粒体通透性转变（MPT）。我们的中心假设是：增加的线粒体Ca2+浓度（[Ca2+]m）有利于线粒体裂变动力学的平衡，从而增加ROS的产生。由此产生的氧化环境导致额外的线粒体Ca2+内流。[Ca2+]m和ROS的增加都增加了MPT打开的可能性，进一步增加了ROS的产生。最终，这个正反馈回路被Ca2+和ROS激活的线粒体Ca2+外排机制抵消，包括Na/Ca交换和MPT。三个具体目的是：1)确定增加的[Ca2+]m是否促进线粒体裂变过程，从而导致ROS生成增加。2)评估含有CypD的MPT通路在[Ca2+]m介导的ROS生成中的作用。3)确定MPT作为线粒体Ca2+快速外排机制的作用。

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.