Role of mitochondria-associated spaces in the regulation of compartmentation of cAMP signaling

线粒体相关空间在 cAMP 信号传导调节中的作用

基本信息

批准号：
10077909
负责人：
Shailesh Agarwal
金额：
$ 20.77万
依托单位：
UNIVERSITY OF NEVADA RENO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10077909
关键词：
A kinase anchoring protein Adopted Affect Anatomy Arrhythmia Biosensor Buffers Cardiac Cardiac Myocytes Cardiovascular Diseases Cardiovascular Physiology Cardiovascular system Cell Volumes Cells Centers of Research Excellence Computer Models Computing Methodologies Confocal Microscopy Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cytoskeletal Proteins Cytosol Development Diffuse Diffusion Disease Enzymes Event Fluorescence Resonance Energy Transfer G-Protein-Coupled Receptors Goals Health Heart Heart Diseases Heart Hypertrophy Heart failure Human Hypertrophy Image Individual Lead Link Location Mathematics Measures Mediating Membrane Membrane Microdomains Microtubules Mitochondria Molecular Morphology Movement Muscle Cells Myofibrils Nevada Organelles Outcome Play Positioning Attribute Process Production Prostaglandin Receptor Protein Sortings Proteins Regulation Role Sarcoplasmic Reticulum Second Messenger Systems Signal Transduction Spectrum Analysis Structure Study models Subcellular Spaces Techniques Testing Therapeutic Ventricular base beta-adrenergic receptor computer studies enzyme activity glucose-regulated proteins heart function knock-down mathematical model muscle LIM protein novel therapeutic intervention overexpression patch clamp phosphoric diester hydrolase prevent protein expression receptor response segregation small hairpin RNA solute

项目摘要

Project Summary Various G-protein-coupled receptors elicit distinct functional responses within a cell, even though they use the common diffusible second messenger cAMP. For instance, while stimulation of either β-adrenergic receptors or E-type prostaglandin receptors leads to cAMP production, only β-adrenergic receptors regulate cardiac myocyte contractility. The ability of a cell to distinguish between cAMP produced in the same cell can only be explained if engagement of different receptors generates distinct receptor-specific pools of cAMP. However, the underlying mechanisms responsible for creating compartmentalized cAMP are not completely understood. Compartmentalized cAMP signaling regulates cardiac contractility and thus is essential for normal functioning of the heart. Consistent with this, dysregulation of cAMP compartmentalization has been linked to several cardiovascular diseases, including cardiac arrhythmias, hypertrophy, and heart failure. Most previous studies have focused on activities of phosphodiesterases, the enzymes that breakdown cAMP, to explain cAMP compartmentation. However, several mathematical studies have predicted that PDE activity alone is not sufficient. These studies have suggested that the mobility of cAMP must be slower than free diffusion to prevent cAMP from reaching non-specific target proteins. We have recently demonstrated that the intracellular mobility of cAMP is markedly hampered by buffering mediated by mitochondria-associated protein kinase A. Now, a new computational study has predicted that, in addition to slow diffusion of cAMP, anatomically restricted spaces within a cell are key to hindering cAMP movement. In cardiac myocytes, mitochondria occupy 30% of the cell volume and are associated with constrained spaces through interactions with the sarcoplasmic reticulum and cytoskeletal proteins. The overall aim of this proposal is to explore the concept that the tight spaces associated with mitochondria regulate cAMP compartmentation. The tethering of mitochondria to the sarcoplasmic reticulum by the proteins, mitofusin-2 (MFN2), glucose-regulated protein 75 (GRP75), and phosphofurin acidic cluster sorting protein 2 (PACS2), creates tight spaces between these organelles. In the FIRST AIM of this study, we will test the hypothesis that the anatomically restricted spaces between mitochondria and the sarcoplasmic reticulum hinder cAMP movement and contribute to cAMP compartmentation. In cardiac myocytes, mitochondrial arrangement is regulated by microtubules and muscle LIM protein (MLP). Disruption of microtubules or MLP causes disorganization of mitochondria and alters mitochondrial morphology, thereby changing the cytosolic spaces associated with mitochondria. Thus, in the SECOND AIM, we hypothesize that cAMP compartmentation is hampered following mitochondrial derangement in microtubule-disrupted cells. To test these hypotheses, we adopt multipronged and complementary approaches to study cAMP compartmentation. Using a variety of advanced techniques, we will measure cAMP mobility, changes in cAMP levels within specific intracellular locations, changes in Ca2+ channel currents and intracellular Ca2+ transients, and test changes in functional responses, such as cell shortening, following stimulation of individual receptors. The goal of this proposal is to elucidate the fundamental mechanisms responsible for facilitating cAMP compartmentation. We believe that this approach may ultimately lead to the development of potential therapeutic strategies to overcome the burden of cardiac diseases.

项目概要各种 G 蛋白偶联受体在细胞内引发不同的功能反应，尽管它们使用例如，当刺激 β-肾上腺素受体或 E型前列腺素受体导致cAMP产生，只有β-肾上腺素能受体调节心肌细胞细胞区分同一细胞中产生的 cAMP 的能力只能在以下情况下得到解释：不同受体的结合会产生不同的受体特异性 cAMP 库。负责产生区室化 cAMP 的机制尚不完全清楚。区室化的 cAMP 信号传导调节心肌收缩力，因此对于心脏的正常功能至关重要与此一致的是，cAMP 区室化的失调与多种因素有关。心血管疾病，包括心律失常、肥厚和心力衰竭。专注于磷酸二酯酶（分解 cAMP 的酶）的活性，以解释 cAMP 然而，一些数学研究预测 PDE 活性本身并不能。这些研究表明，cAMP 的迁移率必须慢于自由扩散才能防止。我们最近证明了 cAMP 能够到达非特异性靶蛋白。 cAMP 的释放受到线粒体相关蛋白激酶 A 介导的缓冲作用的显着阻碍。现在，一种新的方法计算研究预测，除了 cAMP 的缓慢扩散之外，解剖学上受限的空间细胞内的线粒体是阻碍 cAMP 运动的关键。在心肌细胞中，线粒体占据了细胞的 30%。体积并通过与肌浆网的相互作用与受限空间相关联该提案的总体目标是探索紧密空间相关的概念。与线粒体一起调节 cAMP 区室线粒体与肌浆网的束缚。由蛋白质 mitofusin-2 (MFN2)、葡萄糖调节蛋白 75 (GRP75) 和磷酸呋喃酸性簇组成分选蛋白 2 (PACS2) 在这些细胞器之间创造了紧密的空间。将检验线粒体和肌浆之间的解剖学限制空间的假设网状结构阻碍 cAMP 运动并有助于心肌细胞、线粒体中的 cAMP 区室化。排列受微管和肌肉 LIM 蛋白 (MLP) 的破坏。导致线粒体解体并改变线粒体形态，从而改变细胞质因此，在第二个目标中，我们勇敢地面对 cAMP 区室。微管破坏的细胞中线粒体紊乱后，线粒体功能受到阻碍。为了检验这些假设，我们进行了实验。采用多管齐下、互补的方法来研究 cAMP 区室。先进的技术，我们将测量cAMP的流动性，特定细胞内cAMP水平的变化位置、Ca2+ 通道电流和细胞内 Ca2+ 瞬变的变化，并测试功能的变化该提案的目标是刺激个体受体后的反应，例如细胞缩短。阐明了促进 cAMP 区室化的基本机制。方法可能最终导致开发潜在的治疗策略来克服负担心脏病。