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The Complexities of cAMP Signaling in the Heart
AuthorRudokas, Michael William
AdvisorHarvey, Robert D
Cell and Molecular Pharmacology and Physiology
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The autonomic nervous system regulates heart function to meet the metabolic demands of the body. The sympathetic branch of the autonomic nervous system provides an increase in ventricular force of contraction and rate of relaxation. These changes in contractility occur through the activation of cardiac adrenergic receptors (ARs), which can be divided into two types: (ARs) or (ARs). ARs, the more prominent ARs in the heart, utilize the diffusible second messenger molecule 3',5'-cyclic adenosine monophosphate (cAMP) to translate sympathetic stimulation to changes in cardiac functional properties. 1ARs, which is the only AR subtype expressed in the heart, also produce small functional changes and cardioprotective effects in the case of heart disease. Traditionally, 1ARs signal through a different modality from ARs. However, 1AR stimulation has been previously shown to inhibit AR cardiac functional changes. The first part of this dissertation presents an investigation into whether 1ARs can also regulate cAMP through a non-canonical signaling pathway. The second portion of this dissertation delves deeper into the mechanisms of AR subtype (1AR or 2AR) regulation of cAMP signaling. I hypothesized that intracellularly segregated cAMP microdomains allow for the unique set of functional effects seen from selective stimulation of each AR subtype. To answer these questions, I utilized cAMP sensitive fluorescence resonance energy transfer (FRET)-based biosensors with live cell imaging techniques. Using the non-targeted cytosolically expressed Epac2-camps FRET biosensor, I was able to demonstrate that 1ARs can indeed control basal and AR induced cAMP levels through a tyrosine kinase mediated pathway that works at the level of the AR. Furthermore, I characterized a novel FRET biosensor, Epac2-KAP, which is targeted to the non-junctional sarcoplasmic reticulum. The application of the two biosensors allowed for the confirmation of a compartmentalized 2AR cAMP signal due in part to the activity of phosphodiesterase types 2 and 3. The findings documented in this dissertation provide important advancements in the understanding of the regulation of cardiac cAMP signaling through receptor and compartmentation mechanics. Leveraging these discoveries could lead to a better understanding of heart disease and the possible development of therapeutic treatments.