Abstract
Förster resonance energy transfer (FRET)-based biosensors are powerful tools for real-time monitoring of signaling events in intact cells using fluorescence microscopy. Here, we describe a highly sensitive method which allows FRET-based measurements of the second messenger cGMP in adult mouse ventricular myocytes. Such measurements have been challenging before, primarily due to relatively low cGMP concentrations in cardiomyocytes and limited sensitivity of the available biosensors. With our new technique, one can reliably measure dynamic changes in cGMP upon stimulation of myocytes with natriuretic peptides and other physiological and pharmacological ligands.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Castro LR, Schittl J, Fischmeister R (2010) Feedback control through cGMP-dependent protein kinase contributes to differential regulation and compartmentation of cGMP in rat cardiac myocytes. Circ Res 107:1232–1240
Fischmeister R, Castro LR, Abi-Gerges A, Rochais F, Jurevicius J, Leroy J, Vandecasteele G (2006) Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases. Circ Res 99:816–828
Castro LR, Verde I, Cooper DM, Fischmeister R (2006) Cyclic guanosine monophosphate compartmentation in rat cardiac myocytes. Circulation 113:2221–2228
Nikolaev VO, Lohse MJ (2009) Novel techniques for real-time monitoring of cGMP in living cells. Handb Exp Pharmacol 191:229–243
Honda A, Adams SR, Sawyer CL, Lev-Ram V, Tsien RY, Dostmann WR (2001) Spatiotemporal dynamics of guanosine 3′,5′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc Natl Acad Sci USA 98:2437–2442
Russwurm M, Mullershausen F, Friebe A, Jager R, Russwurm C, Koesling D (2007) Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach. Biochem J 407:69–77
Sato M, Hida N, Ozawa T, Umezawa Y (2000) Fluorescent indicators for cyclic GMP based on cyclic GMP-dependent protein kinase Ialpha and green fluorescent proteins. Anal Chem 72:5918–5924
Nausch LW, Ledoux J, Bonev AD, Nelson MT, Dostmann WR (2008) Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors. Proc Natl Acad Sci USA 105:365–370
Nikolaev VO, Gambaryan S, Lohse MJ (2006) Fluorescent sensors for rapid monitoring of intracellular cGMP. Nat Methods 3:23–25
Stangherlin A, Gesellchen F, Zoccarato A, Terrin A, Fields LA, Berrera M, Surdo NC, Craig MA, Smith G, Hamilton G et al (2011) cGMP signals modulate cAMP levels in a compartment-specific manner to regulate catecholamine-dependent signaling in cardiac myocytes. Circ Res 108:929–939
Mongillo M, Tocchetti CG, Terrin A, Lissandron V, Cheung YF, Dostmann WR, Pozzan T, Kass DA, Paolocci N, Houslay MD et al (2006) Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway. Circ Res 98:226–234
Niino Y, Hotta K, Oka K (2009) Simultaneous live cell imaging using dual FRET sensors with a single excitation light. PLoS One 4:e6036
Börner S, Schwede F, Schlipp A, Berisha F, Calebiro D, Lohse MJ, Nikolaev VO (2011) FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells. Nat Protoc 6:427–438
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (grant NI 1301/1-1 and SFB 1002 to V.O.N) and University of Göttingen Medical Center (“pro futura” grant to V.O.N.).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Götz, K.R., Nikolaev, V.O. (2013). Advances and Techniques to Measure cGMP in Intact Cardiomyocytes. In: Krieg, T., Lukowski, R. (eds) Guanylate Cyclase and Cyclic GMP. Methods in Molecular Biology, vol 1020. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-459-3_7
Download citation
DOI: https://doi.org/10.1007/978-1-62703-459-3_7
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-458-6
Online ISBN: 978-1-62703-459-3
eBook Packages: Springer Protocols