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Ca 2+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脏功能的生理和病理。线粒体Ca ~（2+）和ROS在介导心肌细胞的生与死中的关键作用已被广泛认识。钙离子介导的生命与死亡之间的分离在于Ca 2+和ROS浓度之间的最佳平衡。该领域的大多数现有研究分别集中在Ca 2+或ROS稳态。然而，这两种信号通路之间的相互作用刚刚开始受到少数实验室的关注。有趣的是，有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，Ca 2+和ROS稳态的生理调节中起着至关重要的作用。在这个提议中，我们将研究这三个重要的组成部分（Ca 2+，ROS和线粒体裂变机制）如何沟通，以调节心脏Ca 2+和ROS串扰信号。我们将使用多学科的方法，包括细胞生物学技术（例如共聚焦显微镜）、分子生物学（例如基因转移）、生物化学（例如蛋白质印迹法），和转基因小鼠模型（例如亲环素D（CypD）敲除小鼠和mt-cpYFP转基因小鼠）来阐明ROS和Ca 2+的机制共生与线粒体分裂蛋白DLP 1和线粒体通透性转换（MPT）的独特重点。我们的中心假设是：增加的线粒体Ca 2+浓度（[Ca 2 +]m）有利于线粒体动力学向分裂的平衡，这又增加了ROS的产生。由此产生的氧化环境导致额外的线粒体Ca 2+内流。[Ca 2 +]m和ROS的增加都增强了MPT的开放概率，从而进一步增加了ROS的产生。最终，该正反馈回路被Ca 2+和ROS激活的线粒体Ca 2+流出机制（包括Na/Ca交换和MPT）抵消。这三个具体目标是：1）确定增加的[Ca 2 +]m是否促进线粒体分裂过程，然后导致ROS产生增加。2)评估含有CypD的MPT途径在[Ca 2 +] m介导的ROS产生中的作用。3)确定MPT作为线粒体快速Ca 2+流出机制的作用。 公共卫生相关性：氧化应激可引起许多人类疾病，包括慢性心力衰竭中的心肌病、缺血性心脏病、神经退行性疾病、糖尿病、肥胖和衰老。拟议的研究重点是阐明活性氧产生的细胞机制。我们的目标不仅是对氧化应激介导的疾病机制做出科学贡献，而且还为治疗这些使人衰弱的疾病开发可能的治疗手段。

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.