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Integration and Compartmentation of β-Adrenergic Signaling in Cardiomyocytes
We and others have demonstrated that cardiomyocytes contain distinct cAMP nanodomains that restrict cAMP production and elevation to specific intracellular nanodomains. This is achieved through localized production by adenylyl cyclases and limited free diffusion of cAMP, mediated by buffering and degradation via phosphodiesterases. Recently, this concept has been extended to include β-adrenergic receptors (β-ARs), which are now recognized as integral components of these nanodomains, spatially confining the activation of adenylyl cyclases.
Our central hypothesis is that distinct β-AR pools play a critical role in regulating individual cAMP nanodomains. We aim to systematically investigate the regulation of these β-AR/cAMP nanodomains and their impact on cardiomyocyte contractile function, which remains poorly understood. This research will provide insights into how GPCR stimulation is integrated and compartmentalized within cells, aligning synergistically with the objectives of FOR 5807. To achieve this, we will first precisely map the localization of β1- and β2-AR subtypes, not only on the plasma membrane but also at specific intracellular sites. These include key players in excitation-contraction coupling and intracellular compartments such as mitochondria and nuclei. Using self-labeling tagging strategies for high-resolution imaging, we will pinpoint their distribution and identify the trafficking sequences responsible for their compartment-specific expression by selectively deleting regions of the N- and C-terminal tails of the receptors. We will further integrate functional analyses by combining contraction studies in human-induced pluripotent stem cell-derived cardiomyocytes, adult mouse and rat cardiomyocytes, and force measurements in ventricular slices from mice and humans. These will be paired with nanodomain-specific cAMP imaging. To discriminate between plasmalemmal and intracellular β-AR pools, we will employ cell-membrane-permeable and impermeable agonists and antagonists, and we will use the optoGPCR JellyOP targeted to specific nanodomains and compartments for domain-specific stimulation. This will allow us to identify the unique roles of individual nanodomains and their network-level interactions. Finally, we will develop all-optical approaches to determine the conditions under which cAMP increases remain localized to a single nanodomain or spread to others.
In summary, this study will elucidate the roles of localized β-AR pools within cAMP nanodomains, the extent of compartmentation in cardiomyocytes, and the functional implications for contractility regulation. This systematic approach will provide a detailed map of the intracellular cAMP network and its organization. In a subsequent funding phase, we will expand these investigations to disease models, including hypertrophy and heart failure and explore how disruptions in this network contribute to cardiac arrhythmias.
