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

项目概要/摘要 产生单独的cAMP细胞内池允许各种G蛋白偶联受体引起不同的细胞内cAMP水平。 在同一个细胞中的功能反应。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的产生，只有β-肾上腺素能受体调节心肌细胞 收缩性cAMP区室化的失调与几种心血管疾病有关， 包括心律失常、肥大和心力衰竭。然而，负责的潜在机制 产生区室化cAMP的机制还不完全清楚。以前的研究主要集中在 磷酸二酯酶（分解cAMP的酶）的活性，以解释cAMP区室化。 然而，一些数学研究已经预测，PDE活性本身是不够的。这些研究 已经提出cAMP的流动性必须比自由扩散慢，以防止cAMP到达 非特异性靶蛋白。我们最近证明，cAMP的细胞内流动性是显着的， 通过由与蛋白激酶A相关的蛋白激酶A介导的缓冲而阻碍。现在，一项新的计算研究 预测，除了缓慢的扩散，由解剖学限制的空间所施加的物理障碍 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的空间 proteins.本提案的总体目标是探讨与之相关的紧凑空间的概念， 线粒体调节cAMP区室化。葡萄糖调节蛋白75（GRP 75）和肌肉LIM蛋白 (MLP)已经显示出调节周围SR之间的线粒体的紧密排列， 肌原纤维此外，以前的研究表明，线粒体和线粒体之间的空间明显扩大， 衰竭的心室肌细胞的邻近结构。在本研究的第一个目的中，我们将测试 假设GRP 75诱导线粒体和肌浆网之间空间收紧 阻碍cAMP运动并促进cAMP区室化。在第二个目标中，我们将确定 MLP介导的线粒体细胞内排列调节cAMP区室化。在第三个目标中， 我们将检验cAMP信号传导的受损区室化是由于去除了 由于线粒体和相邻细胞器之间的差距变宽而导致的阻塞， 肌细胞为了实现这些目标，我们将采取多管齐下和互补的方法来研究cAMP 分隔使用各种先进的技术，我们将测量cAMP迁移率，受体- 介导的特定细胞内位置的区室化cAMP反应，Ca 2+通道电流， 细胞内Ca 2+瞬变和细胞缩短。本提案的目的是阐明 负责促进cAMP区室化的机制。我们认为，这种方法最终将 导致开发新的治疗策略，以克服人类心脏疾病的负担。

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.