A junctional cAMP compartment regulates rapid Ca signaling in atrial myocytes.

Tobias Kohl, Stephan E Lehnart, Viacheslav O Nikolaev, Niels Voigt, Gerd Hasenfuß, Leonardo Sacconi, Despina Sanoudou, Demetrios A Arvanitis, Jörg W Wegener, Sören Brandenburg, Saskia I Amlaz, Mufassra Mushtaq, Marina Scardigli, Dennis Uhlenkamp, Hariharan Subramanian, Vanessa Steckmeister, Jan Pawlowitz

Journal: Journal of molecular and cellular cardiology 2022;165():141-157

PMID: 35033544

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Abstract

Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca-induced Ca release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following β-adrenergic stimulation. Here, we hypothesized that the baseline synthesis of 3',5'-cyclic adenosine monophosphate (cAMP) is constitutively augmented in the axial tubule junctional compartments of atrial myocytes. Confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to RyR2 in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions. Interestingly, a new transgenic junctin-targeted FRET cAMP biosensor was exclusively co-clustered in the junctional compartment, and hence allowed to monitor cAMP selectively in the vicinity of junctional RyR2 channels. To dissect local cAMP levels at axial tubule junctions versus subsurface Ca release sites, we developed a confocal FRET imaging technique for living atrial myocytes. A constitutively high adenylyl cyclase activity sustained increased local cAMP levels at axial tubule junctions, whereas β-adrenergic stimulation overcame this cAMP compartmentation resulting in additional phosphorylation of non-junctional RyR2 clusters. Adenylyl cyclase inhibition, however, abolished the junctional RyR2 phosphorylation and decreased L-type Ca channel currents, while FRET imaging showed a rapid cAMP decrease. In conclusion, FRET biosensor imaging identified compartmentalized, constitutively augmented cAMP levels in junctional dyads, driving both the locally increased phosphorylation of RyR2 clusters and larger L-type Ca current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca-induced Ca release, whereas β-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters.

Address: Cellular Biophysics and Translational Cardiology Section, Department of Cardiology & Pneumology, Heart Research Center Göttingen, University Medical Center Göttingen, Göttingen, Germany; DZHK (German Centre for Cardiovascular Research), partner site Göttingen, Germany; Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Germany. Electronic address: [email protected].; Cellular Biophysics and Translational Cardiology Section, Department of Cardiology & Pneumology, Heart Research Center Göttingen, University Medical Center Göttingen, Göttingen, Germany.; Institute of Pharmacology and Toxicology, Heart Research Center Göttingen, University Medical Center Göttingen, Göttingen, Germany.; Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.; Department of Physics and Astronomy, University of Florence, Florence, Italy; European Laboratory for Non-Linear Spectroscopy and National Institute of Optics (INO-CNR), Sesto Fiorentino, Italy.; Cellular Biophysics and Translational Cardiology Section, Department of Cardiology & Pneumology, Heart Research Center Göttingen, University Medical Center Göttingen, Göttingen, Germany; Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Germany.; Molecular Biology Division, Biomedical Research Foundation, Academy of Athens, Athens, Greece.; European Laboratory for Non-Linear Spectroscopy and National Institute of Optics (INO-CNR), Sesto Fiorentino, Italy; Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg, Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany.; Cellular Biophysics and Translational Cardiology Section, Department of Cardiology & Pneumology, Heart Research Center Göttingen, University Medical Center Göttingen, Göttingen, Germany; DZHK (German Centre for Cardiovascular Research), partner site Göttingen, Germany; Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Germany.; DZHK (German Centre for Cardiovascular Research), partner site Göttingen, Germany; Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Germany; Institute of Pharmacology and Toxicology, Heart Research Center Göttingen, University Medical Center Göttingen, Göttingen, Germany.; Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Germany.; Cellular Biophysics and Translational Cardiology Section, Department of Cardiology & Pneumology, Heart Research Center Göttingen, University Medical Center Göttingen, Göttingen, Germany; DZHK (German Centre for Cardiovascular Research), partner site Göttingen, Germany; Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Germany; BioMET, Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, MD, USA. Electronic address: [email protected].

DOI: 10.1016/j.yjmcc.2022.01.003

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MeSH Terms: Adenylyl Cyclases, Adrenergic Agents, Calcium, Calcium Signaling, Cyclic AMP, Myocytes, Cardiac, Ryanodine Receptor Calcium Release Channel, Sarcoplasmic Reticulum