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：
8761519
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/8761519
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 Heart Heart Diseases Heart failure Homeostasis Human Hydrogen Peroxide In Situ In Vitro Infusion procedures Injury Knock-in Mouse Knock-out Lead Light Lipid Bilayers Mass Spectrum Analysis Metabolic Diseases Mitochondria Modeling Molecular Molecular Biology Morphology Mus Myocardial Ischemia Neurodegenerative Disorders Oxidation-Reduction Oxidative Stress 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 protein tyrosine kinase PYK2 public health relevance 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。在心肌细胞中，已经提出了线粒体Ca2+浓度（[CA2+] M）的增加可增强ATP和ROS的产生以及线粒体裂变。然而，线粒体Ca2+ Uniporter（MTCU）的精确贡献，即线粒体Ca2+涌入的主要机制，在调节线粒体ATP，ROS和裂变方面仍然不确定，主要是由于缺乏其分子同一性。此外，在没有分子信息的情况下，我一直在研究如何在生理和病理条件下调节MTCU的分子机制。 2011年，两项开创性的研究阐明了MTCU复合物的分子成分，包括孔形成单元（MCU），含有螺旋芯结构域的蛋白109a（CCDC109A）和调节成分（MICU1-3）。同时，人们对Ca2+依赖性氧化还原敏感的脯氨酸激酶2（PYK2）充当参与病理心脏重塑和心脏衰竭进展（HF）的关键应激刺激的关键传感器（HF）。有趣的是，从人类和小鼠样品的质谱分析中报道了CCDC109A的基底酪氨酸磷酸化。最后，线粒体Ca2+过载可以通过与线粒体通透性过渡孔（MPTP）开放有关的事件（例如氧化应激和能量耗竭）引起HF。我们假设PYK2磷酸化MCU，从而增加了通过低聚的四聚通道的数量，从而增强了线粒体Ca2+摄取。 [Ca2+] M的增加增加了ROS的产生。 ROS的增加促进了线粒体裂变。从生理上讲，线粒体Ca2+和裂变协同工作有效地增加了ATP的产生。然而，在压力下，过度的PYK2和MCU激活导致病理上高水平的线粒体Ca2+，裂变和ROS，这会导致MPTP延长开放，从而导致细胞损伤/死亡以及随后的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 （超声心动图）和HF的苯肾上腺输注小鼠模型，以获得实验结果，从而导致机械洞察力。通过PYK2来指出MCU的精确磷酸化位点的特征，并通过MCU低聚是独特的。分子机制的阐明如何增加[Ca2+] M诱导裂变将显着增加有关线粒体形式和功能之间串扰信号的新见解。最后，调整PYK2/MCU信号通路治疗人类疾病的前景会令人鼓舞，因为破坏线粒体Ca2+稳态是导致线粒体功能障碍相关的临床疾病表型，包括心脏疾病（包括心脏疾病）（例如HF）（例如HF）糖尿病性残疾人（糖尿病）的关键因素（包括心脏疾病）。