Ca2+ and ROS Crosstalk Signaling in Cardiac Mitochondria

描述(由申请人提供)：线粒体钙离子、活性氧物种(ROS)和形态在控制细胞命运中的关键作用得到了很好的认识。在心肌细胞中，线粒体Ca~(2+)浓度([Ca~(2+)]m)的增加促进了ATP和ROS的生成以及线粒体的分裂。然而，作为线粒体钙离子内流的主要机制，线粒体钙离子单一转运体(MTCU)在调节线粒体ATP、ROS和分裂中的确切作用仍不确定，这主要是因为它缺乏分子同源性。此外，在没有分子信息的情况下，我一直在挑战着研究MTCU在生理和病理条件下如何调节的分子机制。2011年，两项开创性的研究阐明了MTCU复合体的分子组成，包括孔形成单位(MCU)、含有卷曲结构域的蛋白109A(CCDC109A)和调节成分(MICU1-3)。同时，钙离子依赖的氧化还原敏感的富含脯氨酸的酪氨酸激酶2(PYK2)在病理性心脏重塑和心力衰竭(HF)的进展中作为应激刺激的关键转导而发挥作用。有趣的是，CCDC109A的基础酪氨酸磷酸化是从人和小鼠样本的质谱分析中报道的。最后，线粒体钙超载可通过与线粒体通透性转换孔(MPTP)开放相关的事件(如氧化应激和能量耗竭)引起心衰。我们假设，Pyk2使MCU磷酸化，从而通过寡聚增加了四倍体通道的数量，从而增强了线粒体对钙的摄取。[Ca~(2+)]_m的增加增加了ROS的产生。这种ROS的增加促进了线粒体的分裂。从生理上讲，线粒体Ca~(2+)和分裂协同作用，有效地增加了ATP的产生。然而，在应激状态下，过量的Pyk2和MCU激活导致线粒体钙离子、分裂和ROS的病理性高水平，从而导致MPTP开放时间延长，导致细胞损伤/死亡和随后的心衰。为了验证这一假设，我们将使用多种技术，包括生物化学(从体外到原位分析)，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(带有脂质双层或有丝分裂体的单通道记录)，心脏生理学(超声心动图)，以及苯肾上腺素注射小鼠的HF模型，以获得将导致机理洞察的实验结果。用PYK2精确定位MCU的磷酸化位点，并证明通过MCU齐聚形成功能性的钙离子通透通道是独一无二的。阐明[Ca2+]m增加如何导致分裂的分子机制将显著增加对线粒体形态和功能之间的串扰信号的新见解。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，因为线粒体钙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(如心衰)、神经退行性疾病、代谢性疾病(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.