Ca2+ and ROS Crosstalk Signaling in Cardiac Mitochondria

心脏线粒体中的 Ca2 和 ROS 串扰信号传导

基本信息

批准号：
9037698
负责人：
Shey-Shing Sheu
金额：
$ 38.75万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9037698
关键词：
Aging Animal Model Biochemistry Biological Assay Biophysics Buffers Calcium Cardiac Cardiac Myocytes Carrier Proteins Cell Death Cell Fate Control Cells Cellular biology Cessation of life Chronic Coiled-Coil Domain Complex Cytosol Diabetes Mellitus Diffuse Disease Dynamin Echocardiography Electron Microscopy Elements Environment Event Failure Feedback Fluorescence Resonance Energy Transfer Functional disorder Generations Genes Goals Health Heart Diseases Heart failure Homeostasis Human Hydrogen Peroxide In Situ In Vitro Infusion procedures Injury Knock-in Knock-out Lead Light Lipid Bilayers Mass Spectrum Analysis Metabolic Diseases Mitochondria Modeling Molecular Molecular Biology Morphology Mus Myocardial Ischemia Myocardial dysfunction Neurodegenerative Disorders Oxidation-Reduction Oxidative Stress PTK2B gene Pathology Phenylephrine Phosphorylation Phosphorylation Site Physiological Physiology Post-Translational Protein Processing Production Proteins RNA Interference Reactive Oxygen Species Regulation Reporting Research Research Project Grants Role Sampling Sarcoplasmic Reticulum Signal Pathway Signal Transduction Stimulus Stress Techniques Testing Therapeutic Transducers Tyrosine Phosphorylation Work cell injury clinical phenotype heart cell human disease in vivo insight mitochondrial dysfunction mitochondrial permeability transition pore mouse model novel overexpression sudden cardiac death uptake

项目摘要

DESCRIPTION (provided by applicant): The pivotal role of mitochondrial Ca2+, reactive oxygen species (ROS), and morphology in controlling cell fate is well recognized. In cardiac muscle cells, it has been proposed that increases in mitochondrial Ca2+ concentrations ([Ca2+]m) enhance ATP and ROS generation as well as mitochondrial fission. However, the precise contribution of mitochondrial Ca2+ uniporter (mtCU), the primary mechanism for mitochondrial Ca2+ influx, in regulating mitochondrial ATP, ROS, and fission is still inconclusive mostly due to the lack of its molecular identity. Furthermore, without the molecular information, i has been challenging to study the molecular mechanisms of how mtCU is regulated in the physiological and pathological conditions. In 2011, two ground-breaking studies have elucidated the molecular components of the mtCU complexes including the pore forming unit (MCU), the coiled-coil domain-containing protein 109A (CCDC109A), and regulatory components (MICU1-3). Meanwhile, it has gained appreciation that Ca2+-dependent redox-sensitive proline-rich tyrosine kinase 2 (Pyk2) functions as a key transducer of stress stimuli involved in pathological cardiac remodeling and the progression of heart failure (HF). Intriguingly, basal tyrosine phosphorylation of CCDC109A was reported from mass spectroscopy analyses of human and mouse samples. Finally, mitochondrial Ca2+ overload can cause HF through events (e.g. oxidative stress and energy depletion) associated with the opening of mitochondrial permeability transition pores (mPTP). We hypothesize that Pyk2 phosphorylates MCU that increases the number of tetrametric channels by oligomerization so that mitochondrial Ca2+ uptake is enhanced. The increases in [Ca2+]m augments ROS generation. This increase in ROS promotes mitochondrial fission. Physiologically, mitochondrial Ca2+ and fission work in concert to increase ATP production efficiently. However, under stress, excessive Pyk2 and MCU activation leads to pathologically high levels of mitochondrial Ca2+, fission, and ROS, which cause prolonged mPTP opening, resulting in cell injury/death and subsequent HF. To test this hypothesis, we will employ multiple techniques including biochemistry (from in vitro to in situ assays), molecular biology (gene knock in or knock out, overexpression, RNA interference), cell biology (confocal, fluorescence resonance energy transfer, electron microscopy), biophysics (single channel recordings with lipid bilayer or mitoplast), cardiac physiology (echocardiogram), and phenylephrine infusion mouse model of HF, to obtain experimental results that will lead to mechanistic insights. The feature of pinpointing the precise phosphorylation sites of MCU by Pyk2 and demonstrating the formation of functional Ca2+ permeable channels through MCU oligomerization is unique. The elucidation of molecular mechanisms how increases in [Ca2+]m induce fission will significantly add novel insights regarding crosstalk signaling between mitochondrial form and function. Finally, the prospect of tweaking Pyk2/MCU signaling pathways for treating human diseases will be encouraging because the destruction of mitochondrial Ca2+ homeostasis is a key element for leading to mitochondrial dysfunction-associated clinical phenotypes including heart diseases (e.g. HF), neurodegenerative diseases, metabolic diseases (diabetes), and aging.

描述（由申请人提供）：线粒体 Ca2+、活性氧 (ROS) 和形态在控制细胞命运中的关键作用已得到广泛认可。在心肌细胞中，有人提出线粒体 Ca2+ 浓度 ([Ca2+]m) 的增加会增强 ATP 和 ROS 的生成以及线粒体裂变。然而，线粒体 Ca2+ 单向转运蛋白 (mtCU)（线粒体 Ca2+ 内流的主要机制）在调节线粒体 ATP、ROS 和裂变方面的精确贡献仍然没有定论，主要是由于缺乏其分子特性。此外，在没有分子信息的情况下，我一直难以研究 mtCU 在生理和病理条件下如何调节的分子机制。 2011 年，两项突破性研究阐明了 mtCU 复合物的分子成分，包括孔形成单元 (MCU)、含有卷曲螺旋结构域的蛋白 109A (CCDC109A) 和调节成分 (MICU1-3)。同时，人们认识到 Ca2+ 依赖的氧化还原敏感的富含脯氨酸的酪氨酸激酶 2 (Pyk2) 作为应激刺激的关键传感器，参与病理性心脏重塑和心力衰竭 (HF) 的进展。有趣的是，从人类和小鼠样本的质谱分析中报告了 CCDC109A 的基础酪氨酸磷酸化。最后，线粒体 Ca2+ 超载可通过与线粒体通透性转换孔 (mPTP) 打开相关的事件（例如氧化应激和能量消耗）引起心力衰竭。我们假设 Pyk2 磷酸化 MCU，通过寡聚化增加四长通道的数量，从而增强线粒体 Ca2+ 吸收。 [Ca2+]m 的增加会增加 ROS 的产生。 ROS 的增加促进线粒体裂变。生理上，线粒体 Ca2+ 和裂变协同工作，有效增加 ATP 产量。然而，在压力下，过度的 Pyk2 和 MCU 激活会导致病理性高水平的线粒体 Ca2+、裂变和 ROS，从而导致 mPTP 开放时间延长，导致细胞损伤/死亡以及随后的心力衰竭。为了检验这一假设，我们将采用多种技术，包括生物化学（从体外到原位测定）、分子生物学（基因敲入或敲除、过度表达、RNA干扰）、细胞生物学（共聚焦、荧光共振能量转移、电子显微镜））、生物物理学（脂质双层或线粒体的单通道记录）、心脏生理学（超声心动图）和去氧肾上腺素输注小鼠心力衰竭模型，以获得实验结果，从而导致机制的见解。 Pyk2 能够精确定位 MCU 的磷酸化位点，并通过 MCU 寡聚化证明功能性 Ca2+ 通透通道的形成，其特点是独一无二的。阐明 [Ca2+]m 增加如何诱导裂变的分子机制将显着增加有关线粒体形式和功能之间串扰信号传导的新见解。最后，调整 Pyk2/MCU 信号通路治疗人类疾病的前景将是令人鼓舞的，因为线粒体 Ca2+ 稳态的破坏是导致线粒体功能障碍相关临床表型的关键因素，包括心脏病（例如心力衰竭）、神经退行性疾病、代谢性疾病等。疾病（糖尿病）和衰老。