Abstract 464: Two Different Microdomains of β 1 -adrenoreceptor Signaling Revealed by Live Cell Imaging

Abstract

Background: 3’,5’-cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger and a crucial regulator of cardiac function and disease. In cardiomyocytes, it is produced predominantly after activation of β 1 -adrenergic receptors (β 1 -ARs) by catecholamines and acts intracellularly in discrete functionally relevant microdomains formed, for example, around calcium-handling proteins. Previously, we reported that β 1 -ARs are distributed across various cardiomyocyte membrane areas, including transverse (T)-tubules and cell crests. However, it is unknown whether these two β 1 -AR pools contribute differentially to the regulation of cardiac contractility and gene expression. Methods and Results: To directly visualize receptor-microdomain communication in cardiomyocytes, we established a combination of scanning ion conductance microscopy (SICM) with transgenically expressed targeted Förster resonance energy transfer (FRET)-based biosensors. Using this approach, we measured local cAMP responses in distinct microdomains of mouse ventricular cardiomyocytes (such as plasma membrane, cytosol and nucleus) after localized stimulation of β 1 -AR on different membrane structures of healthy and diseased cardiomyocytes. Using a plasma membrane targeted cAMP biosensor, we found that β 1 -AR stimulation at the crest induced stronger cAMP signals compared to β 1 -AR stimulated in the T-tubuli where cAMP was highly confined by PDE3. This difference was abolished in a pressure overload hypertrophy model due to submembrane redistribution of PDEs. Interestingly, crest β 1 -AR signals could propagate deeper inside the cell, inducing higher nuclear cAMP responses than recorded from receptors stimulated in the T-tubules. Conclusions: in the present study, we have demonstrated that β 1 -ARs located in T-tubuli and cell crests form two differentially regulated cAMP microdomains, each having its typical PDE repertoire and generating distinct second messenger signals. More detailed understanding of these two microdomains at different subsarcolemmal locations may contribute to new therapeutic strategies including more specific β-blockers.