Regulation of compartmentalized cAMP signaling by mitochondria-associated spaces in adult ventricular myocytes

成人心室肌细胞中线粒体相关空间对区室化 cAMP 信号传导的调节

• 批准号: 10645094
• 负责人: Shailesh Agarwal
• 金额: $ 46.2万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10645094
• 关键词: A kinase anchoring protein; Adopted; Adult; Affect; Anatomy; Arrhythmia; Biosensor; Buffers; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cell Volumes; Cells; Computer Models; Computing Methodologies; Confined Spaces; Confocal Microscopy; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Proteins; Development; Diffusion; Disease; Disease model; Electrophysiology (science); Enzymes; Event; Excision; Fluorescence Resonance Energy Transfer; G-Protein-Coupled Receptors; Goals; Heart; Heart Diseases; Heart Hypertrophy; Heart failure; Human; Hypertrophy; Image; Impairment; Individual; Link; Location; Mathematics; Measures; Mediating; Membrane; Membrane Microdomains; Mitochondria; Modeling; Movement; Muscle Cells; Myofibrils; Obstruction; Organelles; Outcome; Play; Positioning Attribute; Process; Production; Prostaglandin Receptor; Proteins; Rattus; Regulation; Role; Sarcoplasmic Reticulum; Second Messenger Systems; Signal Transduction; Spectrum Analysis; Structure; Study models; Subcellular Spaces; Techniques; Testing; Therapeutic; Ventricular; beta-adrenergic receptor; computer studies; enzyme activity; glucose-regulated proteins; heart function; knock-down; mathematical model; muscle LIM protein; nanoscale; novel therapeutic intervention; patch clamp; phosphoric diester hydrolase; prevent; protein expression; receptor; response; restraint; segregation; small hairpin RNA

PROJECT SUMMARY/ABSTRACT Generating separate intracellular pools of cAMP allows various G protein-coupled receptors to elicit distinct functional responses in a same cell. 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. Dysregulation of cAMP compartmentalization has been linked to several cardiovascular diseases, including cardiac arrhythmias, hypertrophy, and heart failure. However, the underlying mechanisms responsible for creating compartmentalized cAMP are not completely understood. 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, physical barriers imposed by anatomically restricted spaces within a cell are key to hindering cAMP movement. In cardiac myocytes, mitochondria occupy 30% of the cell volume, and form 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. Glucose-regulated protein 75 (GRP75) and muscle LIM protein (MLP) have been shown to regulate the compact arrangement of mitochondria between the surrounding SR and myofibrils. Moreover, previous studies have shown a marked widening of the space between mitochondria and the neighboring structures in failing ventricular myocytes. In the FIRST AIM of this study, we will test the hypothesis that GRP75-induced tightening of the space between mitochondria and the sarcoplasmic reticulum hinder cAMP movement and contribute to cAMP compartmentation. In the SECOND AIM, we will determine if MLP-mediated intracellular arrangement of mitochondria regulates cAMP compartmentation. In the THIRD AIM, we will test the hypothesis that the compromised compartmentation of cAMP signaling is due the removal of obstruction as a result of the widening of the gap between mitochondria and adjacent organelles in failing myocytes. To accomplish these aims, we will adopt multipronged and complementary approaches to study cAMP compartmentation. Using a variety of advanced techniques, we will measure changes to cAMP mobility, receptor- mediated compartmentalized cAMP responses within specific intracellular locations, Ca2+ channel currents, intracellular Ca2+ transients, and cell shortening. The goal of this proposal is to elucidate the fundamental mechanisms responsible for facilitating cAMP compartmentation. We believe that this approach will ultimately lead to the development of novel therapeutic strategies to overcome the burden of cardiac diseases in humans.

项目概要/摘要 生成单独的 cAMP 细胞内池允许各种 G 蛋白偶联受体引发不同的 同一细胞内的功能反应。例如，当刺激 β-肾上腺素能受体或 E 型受体时 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 收缩性。 cAMP 区室化失调与多种心血管疾病有关， 包括心律失常、肥厚和心力衰竭。然而，负责的底层机制 对于创建区室化 cAMP 的作用尚不完全清楚。之前的大多数研究都集中在 磷酸二酯酶（分解 cAMP 的酶）的活性，以解释 cAMP 的区室分布。 然而，一些数学研究预测仅 PDE 活性是不够的。这些研究 已经表明，cAMP 的迁移率必须慢于自由扩散，以防止 cAMP 到达 非特异性靶蛋白。我们最近证明 cAMP 的细胞内迁移率显着 受到线粒体相关蛋白激酶 A 介导的缓冲的阻碍。现在，一项新的计算研究 预测，除了缓慢的扩散之外，解剖学上受限的空间造成的物理障碍 细胞内是阻碍 cAMP 运动的关键。在心肌细胞中，线粒体占细胞的30​​% 体积，并通过与肌浆网和细胞骨架的相互作用形成受限空间 蛋白质。该提案的总体目标是探索与狭小空间相关的概念 线粒体调节 cAMP 区室。葡萄糖调节蛋白 75 (GRP75) 和肌肉 LIM 蛋白 (MLP) 已被证明可以调节周围 SR 和 SR 之间线粒体的紧凑排列。 肌原纤维。此外，之前的研究表明线粒体和线粒体之间的空间显着扩大。 衰竭心室肌细胞的邻近结构。在本研究的第一个目标中，我们将测试 假设 GRP75 诱导线粒体和肌浆网之间的空间收紧 阻碍 cAMP 运动并有助于 cAMP 区室化。在第二个目标中，我们将确定是否 MLP 介导的线粒体细胞内排列调节 cAMP 区室。在第三个目标中， 我们将检验以下假设：cAMP 信号传导受损是由于去除了 由于线粒体和相邻细胞器之间的间隙扩大而导致的阻塞 肌细胞。为了实现这些目标，我们将采用多管齐下、互补的方法来研究cAMP 划分。使用各种先进技术，我们将测量 cAMP 迁移率、受体- 介导特定细胞内位置内的区室化 cAMP 反应、Ca2+ 通道电流、 细胞内 Ca2+ 瞬变和细胞缩短。该提案的目标是阐明基本原理 负责促进 cAMP 区室化的机制。我们相信这种方法最终将 导致开发新的治疗策略来克服人类心脏病的负担。