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

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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.