摘要：SummarySinoatrial node (SAN) cells are the pacemakers of the heart. This study describes a method for culturing and infection of adult mouse SAN cells with FRET-based biosensors that can be exploited to examine signaling events. SAN cells cultured in media with blebbistatin or (S)-nitro-blebbistatin retain their morphology, protein distribution, action potential (AP) waveform, and cAMP dynamics for at least 40 h. SAN cells expressing targeted cAMP sensors show distinct β-adrenergic-mediated cAMP pools. Cyclic GMP, protein kinase A, Ca2+/CaM kinase II, and protein kinase D in SAN cells also show unique dynamics to different stimuli. Heart failure SAN cells show a decrease in cAMP and cGMP levels. In summary, a reliable method for maintaining adult mouse SAN cells in culture is presented, which facilitates studies of signaling networks and regulatory mechanisms during physiological and pathological conditions.Graphical abstractDisplay OmittedHighlights•Optimized culturing method of adult SAN cells from healthy and disease-state mice•Cultured adult SAN cells can be infected with diverse fluorescence biosensors•Examination of signaling networks, including discrete cAMP pools, in adult SAN cells•SAN cells cAMP and cGMP signals are impaired during heart failureCell biology; Functional aspects of cell biology; Biology experimental methods
