Deciphering cellular signals in adult mouse sinoatrial node cells
Gopireddy R. Reddy,
Lu Ren,
Phung N. Thai,
Jessica L. Caldwell,
Manuela Zaccolo,
Julie Bossuyt,
Crystal M. Ripplinger,
Yang K. Xiang,
Madeline Nieves-Cintrón,
Nipavan Chiamvimonvat,
Manuel F. Navedo
Affiliations
Gopireddy R. Reddy
Department of Pharmacology, University of California Davis, One Shields Avenue MED: PHARM Tupper 242, Davis, CA 95616, USA
Lu Ren
Department of Internal Medicine, University of California Davis, 451 Health Science Drive, GBSF 6315, Davis, CA 95616, USA
Phung N. Thai
Department of Internal Medicine, University of California Davis, 451 Health Science Drive, GBSF 6315, Davis, CA 95616, USA
Jessica L. Caldwell
Department of Pharmacology, University of California Davis, One Shields Avenue MED: PHARM Tupper 242, Davis, CA 95616, USA
Manuela Zaccolo
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, UK
Julie Bossuyt
Department of Pharmacology, University of California Davis, One Shields Avenue MED: PHARM Tupper 242, Davis, CA 95616, USA
Crystal M. Ripplinger
Department of Pharmacology, University of California Davis, One Shields Avenue MED: PHARM Tupper 242, Davis, CA 95616, USA
Yang K. Xiang
Department of Pharmacology, University of California Davis, One Shields Avenue MED: PHARM Tupper 242, Davis, CA 95616, USA; VA Northern California Healthcare System, 10535 Hospital Way, Mather, CA 95655, USA
Madeline Nieves-Cintrón
Department of Pharmacology, University of California Davis, One Shields Avenue MED: PHARM Tupper 242, Davis, CA 95616, USA
Nipavan Chiamvimonvat
Department of Internal Medicine, University of California Davis, 451 Health Science Drive, GBSF 6315, Davis, CA 95616, USA; VA Northern California Healthcare System, 10535 Hospital Way, Mather, CA 95655, USA; Corresponding author
Manuel F. Navedo
Department of Pharmacology, University of California Davis, One Shields Avenue MED: PHARM Tupper 242, Davis, CA 95616, USA; Corresponding author
Summary: Sinoatrial 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.
