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

成年心室肌细胞线粒体相关空间对区室化 cAMP 信号传导的调节

• 批准号: 10522257
• 负责人: Shailesh Agarwal
• 金额: $ 45.89万
• 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/10522257
• 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; Diffuse; 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; Lead; 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; base; 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; 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.

项目摘要/摘要 产生单独的cAMP细胞内池允许各种G蛋白偶联受体引起独特的 同一细胞中的功能响应。例如，刺激β-肾上腺素受体或E型 前列腺素受体可导致营地产生，只有β-肾上腺素受体调节心肌细胞 收缩力。营地室的失调与几种心血管疾病有关， 包括心律不齐，肥大和心力衰竭。但是，造成的基本机制 为了创建隔间化的营地，尚不完全了解。大多数以前的研究都集中在 磷酸二酯酶的活性，分解营地的酶，以解释营地。 但是，一些数学研究预测，仅PDE活性是不够的。这些研究 已经提出，营地的流动性必须慢于自由扩散以防止营地到达 非特异性靶蛋白。我们最近证明了营地的细胞内移动性明显 通过线粒体相关蛋白激酶A介导的缓冲A的阻碍。现在，一项新的计算研究 已经预测，除了缓慢的扩散外，由解剖限制的空间施加的物理障碍 在牢房内是阻碍营地运动的关键。在心肌细胞中，线粒体占细胞的30​​％ 通过与肌质网和细胞骨架的相互作用，体积和形成了限制的空间 蛋白质。该提案的总体目的是探索与与之相关的紧密空间的概念 线粒体调节营地隔室。葡萄糖调节的蛋白75（GRP75）和肌肉lim蛋白 （MLP）已显示可调节周围SR之间线粒体的紧凑布置 肌原纤维。此外，先前的研究表明，线粒体和 心室肌细胞失败的相邻结构。在这项研究的第一个目的中，我们将测试 假设GRP75引起的线粒体和肌质网之间的空间拧紧 阻碍营地运动，并为营地隔间做出贡献。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营地隔室。在第三个目标中 我们将检验以下假设，即cAMP信号的隔离室被拆除是由于去除的 由于线粒体和相邻细胞器之间的间隙扩大而导致的障碍 心肌细胞。为了实现这些目标，我们将采用多收益和完整的方法来学习训练营 隔室。使用各种高级技术，我们将衡量营地移动性，接收器的变化 - 在特定细胞内位置，Ca2+通道电流中的介导的分室cAMP反应， 细胞内Ca2+瞬变和细胞缩短。该提议的目的是阐明基本 负责支撑营地的机制。我们相信这种方法最终将 导致开发新的治疗策略，以克服人类心脏病的燃烧。