Ca2+ and ROS Crosstalk Signaling in Cardiac Mitochondria

描述（由申请人提供）：线粒体Ca2+、活性氧（ROS）和形态学在控制细胞命运中的关键作用已得到充分认可。在心肌细胞中，已经提出线粒体Ca2+浓度（[Ca2 +] m）的增加增强ATP和ROS产生以及线粒体分裂。然而，线粒体Ca 2+单向转运体（mtCU），线粒体Ca 2+内流的主要机制，在调节线粒体ATP，ROS和裂变的精确贡献仍然是不确定的，主要是由于缺乏其分子身份。此外，在没有分子信息的情况下，研究mtCU在生理和病理条件下如何调节的分子机制一直具有挑战性。2011年，两项突破性的研究阐明了mtCU复合物的分子组成，包括孔形成单元（MCU），含有卷曲螺旋结构域的蛋白质109A（CCDC109A）和调节组分（MICU1 - 3）。与此同时，钙离子依赖性氧化还原敏感性富脯氨酸酪氨酸激酶2（Pyk 2）作为应激刺激的关键转导子参与了病理性心脏重构和心力衰竭（HF）的进展。有趣的是，CCDC109A的基础酪氨酸磷酸化是从人和小鼠样品的质谱分析中报告的。最后，线粒体Ca2+过载可通过与线粒体渗透性转换孔（mPTP）打开相关的事件（例如氧化应激和能量消耗）引起HF。我们假设Pyk2磷酸化MCU，通过寡聚化增加四通道的数量，从而增强线粒体Ca2+摄取。[Ca2 +] m的增加增加了ROS的产生。这种ROS的增加促进线粒体分裂。在生理上，线粒体Ca2+和裂变协同工作，以有效地增加ATP的产生。然而，在应激下，过度的Pyk2和MCU激活导致病理性高水平的线粒体Ca2+、裂变和ROS，这导致延长的mPTP开放，导致细胞损伤/死亡和随后的HF。为了验证这一假设，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（脂质双层或线粒体单通道记录），心脏生理学（超声心动图）和苯丙氨酸输注HF小鼠模型，以获得将导致机理见解的实验结果。Pyk2精确定位MCU的磷酸化位点，并通过MCU寡聚化证明功能性Ca2+渗透通道的形成是独特的。[Ca2 +] m增加如何诱导裂变的分子机制的阐明将显着增加关于线粒体形式和功能之间的串扰信号转导的新见解。最后，调整Pyk2/MCU信号通路用于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HF）、神经退行性疾病、代谢疾病（糖尿病）和衰老。

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.