TY - JOUR
T1 - Deciphering cellular signals in adult mouse sinoatrial node cells
AU - Reddy, Gopireddy R.
AU - Ren, Lu
AU - Thai, Phung N.
AU - Caldwell, Jessica L.
AU - Zaccolo, Manuela
AU - Bossuyt, Julie
AU - Ripplinger, Crystal M.
AU - Xiang, Yang K.
AU - Nieves-Cintrón, Madeline
AU - Chiamvimonvat, Nipavan
AU - Navedo, Manuel F.
N1 - Funding Information:
We thank members of the Navedo lab and Nieves-Cintr?n lab for technical support and for critically reading early versions of the manuscript. We thank Victoria Ramer for help with the generation of the CAMPERCM mouse. We thank Dr. Claudia Moreno and James L. Overton for training on SAN cell isolation technique and technical support, respectively. We thank Drs. Luis F. Santana and Eammon J. Dickson for critically reading an early version of the manuscript. This work was supported by NIH grants R01HL121059, R01HL161872, and R01HL149127, and UC MEXUS-CONACYT CN-19-147 (to M.F.N.), NIH R01 HL085727, R01 HL085844, R01 HL137228, and S10RR033106 (to N.C.), VA Merit Review Grant I01 BX000576 and I01 CX001490 (to N.C.), Postdoctoral Fellowship from NIH T32 Training Grant in Basic & Translational Cardiovascular Science T32HL86350 and F32HL149288 (to P.N.T.), AHA Predoctoral Fellowship Award (to L.R.), British Heart Foundation Programme Grant RG/17/6/32944 (to M.Z.), and AHA Career Development Award 852984 (to M.N.-C.). The contents do not represent the views of the US Department of Veterans Affairs or the United States Government. Conceptualization, G.R.R. L.R. N.C. and M.F.N.; methodology, G.R.R. L.R. P.N.T. J.L.C. J.B. C.M.R. Y.K.X. M,N-C. N.C. and M.F.N.; investigation, G.R.R. L.R. P.N.T. and M.F.N.; formal analysis, G.R.R. L.R. and M.F.N.; resources, J.L.C. M.Z. J.B. C.M.R. Y.K.X. and M.N-C.; writing ? original draft, M.F.N.; writing ? review & editing, G.R.R. L.R. P.N.T. J.L.C. M.Z. J.B. C.M.R. Y.K.X. M.N-C. and N.C.; visualization, M.F.N.; supervision, N.C. and M.F.N.; project administration, N.C. and M.F.N.; funding acquisition, M.Z. M.N-C. N.C. and M.F.N. M.N.-C. is a UC Davis CAMPOS Fellow. N.C. is the Roger Tatarian Endowed Professorship holder in Cardiovascular Medicine and a part-time staff physician at VA Northern California Health Care System, Mather, CA. The authors declare no competing interests. One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in science. One or more of the authors of this paper received support from a program designed to increase minority representation in science.
Funding Information:
We thank members of the Navedo lab and Nieves-Cintrón lab for technical support and for critically reading early versions of the manuscript. We thank Victoria Ramer for help with the generation of the CAMPER CM mouse. We thank Dr. Claudia Moreno and James L. Overton for training on SAN cell isolation technique and technical support, respectively. We thank Drs. Luis F. Santana and Eammon J. Dickson for critically reading an early version of the manuscript. This work was supported by NIH grants R01HL121059 , R01HL161872 , and R01HL149127 , and UC MEXUS-CONACYT CN-19-147 (to M.F.N.), NIH R01 HL085727 , R01 HL085844 , R01 HL137228 , and S10RR033106 (to N.C.), VA Merit Review Grant I01 BX000576 and I01 CX001490 (to N.C.), Postdoctoral Fellowship from NIH T32 Training Grant in Basic & Translational Cardiovascular Science T32HL86350 and F32HL149288 (to P.N.T.), AHA Predoctoral Fellowship Award (to L.R.), British Heart Foundation Programme Grant RG/17/6/32944 (to M.Z.), and AHA Career Development Award 852984 (to M.N.-C.). The contents do not represent the views of the US Department of Veterans Affairs or the United States Government.
Publisher Copyright:
© 2021 The Author(s)
PY - 2022/1/21
Y1 - 2022/1/21
N2 - 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.
AB - 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.
KW - Biology experimental methods
KW - Cell biology
KW - Functional aspects of cell biology
UR - http://www.scopus.com/inward/record.url?scp=85122493111&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85122493111&partnerID=8YFLogxK
U2 - 10.1016/j.isci.2021.103693
DO - 10.1016/j.isci.2021.103693
M3 - Article
AN - SCOPUS:85122493111
VL - 25
JO - iScience
JF - iScience
SN - 2589-0042
IS - 1
M1 - 103693
ER -