Clin Res Cardiol (2025). DOI 10.1007/s00392-025-02737-x
1Universitätsklinikum Hamburg-Eppendorf Experimentelle Herz-Kreislaufforschung Hamburg, Deutschland; 2Universitätsklinikum Hamburg-Eppendorf Institut für Experimentelle Herz-Kreislaufforschung Hamburg, Deutschland; 3Universitätsklinikum Hamburg-Eppendorf Universitäres Herzzentrum Hamburg, Deutschland
BACKGROUND
The Ca2+ ATPase2a (SERCA2a) is the pump responsible for the uptake of Ca2+ into the sarcoplasmic reticulum. Alterations in SERCA2a activity can profoundly affect the ability of the cell to regulate Ca2+ storage and, consequently, its ability to contract or maintain a uniform beat-to-beat pattern. Our aim is to investigate the regulatory pathways that control SERCA2a function, with particular emphasis on the effects of cAMP signaling in this nanodomain.
METHODS
Atrial myocytes were isolated from human atria of patients in sinus rhythm. To investigate compartmentalized cAMP signaling, cardiomyocytes were transduced with adenoviral fluorescence-resonance energy transfer (FRET) biosensors (Epac1-camps, pm-Epac1, E1-JNC, E1-PLB, and TPN-CUTie), targeting key nanodomains: cytosol, plasma membrane, ryanodine receptor, SERCA2a, and troponin. After 48 hours in culture, real-time cAMP dynamics were recorded using live-cell FRET imaging. Cells were exposed to different combinations of a beta-adrenoceptor agonist (Isoprenaline, ISO, 100 nM), a selective PDE8 inhibitor (PF, 30 nM), a non-selective PDE inhibitor (IBMX, 100 μM), a specific activator and an inhibitor of the L-type Ca2+ channel (BayK, 1 mM, and Nifedipine, 50 nM), a ryanodine receptor inhibitor and an agonist (Ryanodine, 200 µM, and Caffeine, 10 mM) . 10 µM forskolin (FSK) was used as positive control. Patch-clamp recordings were conducted to quantify sarcoplasmic reticulum Ca²⁺ load at baseline and following IBMX and ISO treatments, both before and after stimulation protocols.
RESULTS
All nanodomains showed a robust increase in cAMP levels following IBMX treatment. Notably, when PF was added on top of IBMX, cAMP levels in SERCA2a nanodomain were unexpectedly halved. This reduction was also observed when extra Ca²⁺ (1 mM), BayK or caffeine were applied on top of IBMX, indicating a potential inhibitory role of Ca²⁺ on cAMP generation at SERCA2a. Interestingly, blocking Ca²⁺ entry or ryanodine receptors led to increased cAMP levels. Furthermore, FSK stimulation reversed this inhibition. Similar results were obtained upon ISO stimulation. Complementarily, patch-clamp recordings revealed significant differences in sarcoplasmic reticulum Ca²⁺ load across conditions only after the stimulation train.
CONCLUSIONS
Our results suggest a Ca²⁺-dependent regulation of SERCA2a in atrial myocytes isolated from sinus rhythm patients.