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

项目摘要/摘要 产生不同的细胞内cAMP池允许各种G蛋白偶联受体引发不同的 同一细胞中的功能反应。例如，当刺激β-肾上腺素能受体或E-型时 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 伸缩性。CAMP区划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 关于建立隔离营地的问题还没有完全被理解。以前的大多数研究都集中在 磷酸二酯酶的活性，这种酶可以分解cAMP，来解释cAMP的划分。 然而，几项数学研究预测，仅有PDE活动是不够的。这些研究 建议营地的移动速度必须慢于自由扩散，以防止营地到达 非特异性靶蛋白。我们最近证明，cAMP的细胞内迁移率显著 被线粒体相关蛋白激酶A介导的缓冲所阻碍 预测，除了缓慢的扩散，解剖受限的空间造成的物理障碍 在一个细胞内是阻碍营地移动的关键。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 蛋白质。这项提案的总体目标是探索这样一个概念，即狭小的空间与 线粒体调节cAMP的区划。葡萄糖调节蛋白75与肌肉LIM蛋白 (MLP)已被证明调节周围SR和 肌原纤维。此外，先前的研究表明，线粒体和线粒体之间的间隙显著扩大 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 GRP75导致线粒体和肌浆网间隙变窄的假说 阻碍营地的移动，并有助于营地的分隔。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区划。在第三个目标中， 我们将检验这一假设，即cAMP信号的受损区隔是由于移除 衰竭时线粒体与邻近细胞器间隙扩大所致的梗阻 肌细胞。为了达到这些目标，我们将采取多管齐下和相辅相成的方式来开展研习营。 车厢分隔。使用各种先进技术，我们将测量营地流动性、受体- 介导的细胞内特定位置的区间化cAMP反应，钙通道电流， 细胞内钙瞬变和细胞缩短。这项提议的目的是阐明 负责促进营地划分的机制。我们相信，这种做法最终将 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.