Receptor-Cyclic Nucleotide Microdomains in the Heart

  • Nadja I. Bork
  • Viacheslav O. NikolaevEmail author
Part of the Cardiac and Vascular Biology book series (Abbreviated title: Card. vasc. biol., volume 3)


Cyclic nucleotides 3′,5′-cyclic adenosine (cAMP) and 3′,5′-cyclic guanosine monophosphates (cGMP) are important second messengers which regulate cardiac function and disease by acting in spatially separated subcellular microdomains. Function of these microdomains includes but is not limited to the modulation of calcium cycling, excitation-contraction coupling, and cardiac hypertrophy. In recent years, visualization of local compartmentalized cAMP and cGMP dynamics became possible due to rapid development of optical and nonoptical imaging techniques. In this chapter, we will briefly review these state-of-the-art biophysical methods and available fluorescent biosensors which can be used to understand microdomain-specific signaling and its involvement in cardiovascular function and disease.


cAMP cGMP Biosensors FRET Microdomain SICM 



3′,5′-Cyclic adenosine monophosphate




Cyan fluorescent protein


3′,5′-Cyclic guanosine monophosphate


Förster resonance energy transfer


Green fluorescent protein










Scanning ion conductance microscopy


Sarcoplasmic reticulum


Yellow fluorescent protein


β-Adrenergic receptor



The work in authors’ laboratory is supported by the grants from the German Research Foundation (“Deutsche Forschungsgemeinschaft” grants NI 1301/1, NI 1301/2, FOR 2060) and by the Gertraud und Heinz-Rose Stiftung.

Compliance with Ethical Standards

Conflict of Interest Statement

The authors declare that they have no conflict of interest.


  1. Adams SR, Harootunian AT, Buechler YJ et al (1991) Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349:694–697Google Scholar
  2. Allen MD, Zhang J (2006) Subcellular dynamics of protein kinase a activity visualized by FRET-based reporters. Biochem Biophys Res Commun 348:716–721PubMedGoogle Scholar
  3. Ashman DF, Lipton R, Melicow MM et al (1963) Isolation of adenosine 3′, 5′-monophosphate and guanosine 3′, 5′-monophosphate from rat urine. Biochem Biophys Res Commun 11:330–334PubMedGoogle Scholar
  4. Beavo JA, Brunton LL (2002) Cyclic nucleotide research—still expanding after half a century. Nat Rev Mol Cell Biol 3:710–718PubMedPubMedCentralGoogle Scholar
  5. Belge C, Hammond J, Dubois-Deruy E et al (2014) Enhanced expression of beta3-adrenoceptors in cardiac myocytes attenuates neurohormone-induced hypertrophic remodeling through nitric oxide synthase. Circulation 129:451–462PubMedGoogle Scholar
  6. Bers DM (2002) Cardiac excitation-contraction coupling. Nature 415:198–205Google Scholar
  7. Bhargava Y, Hampden-Smith K, Chachlaki K et al (2013) Improved genetically-encoded, FlincG-type fluorescent biosensors for neural cGMP imaging. Front Mol Neurosci 6:26PubMedPubMedCentralGoogle Scholar
  8. Brodde OE, Bruck H, Leineweber K (2006) Cardiac adrenoceptors: physiological and pathophysiological relevance. J Pharmacol Sci 100:323–337PubMedGoogle Scholar
  9. Brooker G, Harper JF, Terasaki WL et al (1979) Radioimmunoassay of cyclic AMP and cyclic GMP. Adv Cyclic Nucleotide Res 10:1–33PubMedGoogle Scholar
  10. Buxton IL, Brunton LL (1983) Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes. J Biol Chem 258:10233–10239Google Scholar
  11. Calebiro D, Nikolaev VO, Gagliani MC et al (2009) Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biol 7:e1000172PubMedPubMedCentralGoogle Scholar
  12. Castro LR, Gervasi N, Guiot E et al (2010) Type 4 phosphodiesterase plays different integrating roles in different cellular domains in pyramidal cortical neurons. J Neurosci 30:6143–6151PubMedPubMedCentralGoogle Scholar
  13. Couto A, Oda S, Nikolaev VO et al (2013) In vivo genetic dissection of O2-evoked cGMP dynamics in a Caenorhabditis elegans gas sensor. Proc Natl Acad Sci U S A 110:E3301–E3310PubMedPubMedCentralGoogle Scholar
  14. De Arcangelis V, Liu R, Soto D et al (2009) Differential association of phosphodiesterase 4D isoforms with beta2-adrenoceptor in cardiac myocytes. J Biol Chem 284:33824–33832PubMedPubMedCentralGoogle Scholar
  15. Depry C, Allen MD, Zhang J (2011) Visualization of PKA activity in plasma membrane microdomains. Mol Biosyst 7:52–58PubMedGoogle Scholar
  16. Di Benedetto G, Zoccarato A, Lissandron V et al (2008) Protein kinase a type I and type II define distinct intracellular signaling compartments. Circ Res 103:836–844Google Scholar
  17. DiPilato LM, Cheng X, Zhang J (2004) Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. Proc Natl Acad Sci U S A 101:16513–16518PubMedPubMedCentralGoogle Scholar
  18. Dyachok O, Isakov Y, Sagetorp J et al (2006) Oscillations of cyclic AMP in hormone-stimulated insulin-secreting beta-cells. Nature 439:349–352PubMedGoogle Scholar
  19. Fischmeister R, Castro LR, Abi-Gerges A et al (2006) Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases. Circ Res 99:816–828Google Scholar
  20. Froese A, Nikolaev VO (2015) Imaging alterations of cardiomyocyte cAMP microdomains in disease. Front Pharmacol 6:172PubMedPubMedCentralGoogle Scholar
  21. Fu Q, Kim S, Soto D et al (2014) A long lasting beta1 adrenergic receptor stimulation of cAMP/protein kinase a (PKA) signal in cardiac myocytes. J Biol Chem 289:14771–14781PubMedPubMedCentralGoogle Scholar
  22. Götz KR, Sprenger JU, Perera RK et al (2014) Transgenic mice for real-time visualization of cGMP in intact adult cardiomyocytes. Circ Res 114:1235–1245PubMedGoogle Scholar
  23. Haj Slimane Z, Bedioune I, Lechene P et al (2014) Control of cytoplasmic and nuclear protein kinase a by phosphodiesterases and phosphatases in cardiac myocytes. Cardiovasc Res 102:97–106PubMedPubMedCentralGoogle Scholar
  24. Hansma PK, Drake B, Marti O et al (1989) The scanning ion-conductance microscope. Science 243:641–643PubMedGoogle Scholar
  25. Hayes JS, Brunton LL, Mayer SE (1980) Selective activation of particulate cAMP-dependent protein kinase by isoproterenol and prostaglandin E1. J Biol Chem 255:5113–5119Google Scholar
  26. Herbst KJ, Coltharp C, Amzel LM et al (2011) Direct activation of Epac by sulfonylurea is isoform selective. Chem Biol 18:243–251PubMedPubMedCentralGoogle Scholar
  27. Herget S, Lohse MJ, Nikolaev VO (2008) Real-time monitoring of phosphodiesterase inhibition in intact cells. Cell Signal 20:1423–1431Google Scholar
  28. Honda A, Adams SR, Sawyer CL et al (2001) Spatiotemporal dynamics of guanosine 3',5'-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc Natl Acad Sci U S A 98:2437–2442PubMedPubMedCentralGoogle Scholar
  29. Klarenbeek J, Goedhart J, van Batenburg A et al (2015) Fourth-generation Epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity. PLoS One 10:e0122513PubMedPubMedCentralGoogle Scholar
  30. Korchev YE, Bashford CL, Milovanovic M et al (1997) Scanning ion conductance microscopy of living cells. Biophys J 73:653–658PubMedPubMedCentralGoogle Scholar
  31. Liu S, Zhang J, Xiang YK (2011) FRET-based direct detection of dynamic protein kinase a activity on the sarcoplasmic reticulum in cardiomyocytes. Biochem Biophys Res Commun 404:581–586PubMedGoogle Scholar
  32. Lohse MJ, Engelhardt S, Eschenhagen T (2003) What is the role of beta-adrenergic signaling in heart failure? Circ Res 93:896–906Google Scholar
  33. Lompre AM, Hajjar RJ, Harding SE et al (2010) Ca2+ cycling and new therapeutic approaches for heart failure. Circulation 121:822–830PubMedPubMedCentralGoogle Scholar
  34. Mehta S, Aye-Han NN, Ganesan A et al (2014) Calmodulin-controlled spatial decoding of oscillatory Ca2+ signals by calcineurin. Elife 3:e03765PubMedPubMedCentralGoogle Scholar
  35. Mohamed TM, Oceandy D, Zi M et al (2011) Plasma membrane calcium pump (PMCA4)-neuronal nitric-oxide synthase complex regulates cardiac contractility through modulation of a compartmentalized cyclic nucleotide microdomain. J Biol Chem 286:41520–41529PubMedPubMedCentralGoogle Scholar
  36. Mongillo M, McSorley T, Evellin S et al (2004) Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases. Circ Res 95:67–75Google Scholar
  37. Mukherjee S, Jansen V, Jikeli JF et al (2016) A novel biosensor to study cAMP dynamics in cilia and flagella. Elife 5Google Scholar
  38. Nausch LW, Ledoux J, Bonev AD et al (2008) Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors. Proc Natl Acad Sci U S A 105:365–370Google Scholar
  39. Niino Y, Hotta K, Oka K (2009) Simultaneous live cell imaging using dual FRET sensors with a single excitation light. PLoS One 4:e6036PubMedPubMedCentralGoogle Scholar
  40. Nikolaev VO, Bunemann M, Hein L et al (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 279:37215–37218Google Scholar
  41. Nikolaev VO, Bunemann M, Schmitteckert E et al (2006a) Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching beta1-adrenergic but locally confined beta2-adrenergic receptor-mediated signaling. Circ Res 99:1084–1091Google Scholar
  42. Nikolaev VO, Gambaryan S, Lohse MJ (2006b) Fluorescent sensors for rapid monitoring of intracellular cGMP. Nat Methods 3:23–25Google Scholar
  43. Nikolaev VO, Moshkov A, Lyon AR et al (2010) Beta2-adrenergic receptor redistribution in heart failure changes cAMP compartmentation. Science 327:1653–1657Google Scholar
  44. Norris RP, Ratzan WJ, Freudzon M et al (2009) Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte. Development 136:1869–1878PubMedPubMedCentralGoogle Scholar
  45. Perera RK, Nikolaev VO (2013) Compartmentation of cAMP signalling in cardiomyocytes in health and disease. Acta Physiol (Oxf) 207:650–662Google Scholar
  46. Perera RK, Sprenger JU, Steinbrecher JH et al (2015) Microdomain switch of cGMP-regulated phosphodiesterases leads to ANP-induced augmentation of beta-adrenoceptor-stimulated contractility in early cardiac hypertrophy. Circ Res 116:1304–1311Google Scholar
  47. Ponsioen B, Zhao J, Riedl J et al (2004) Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator. EMBO Rep 5:1176–1180PubMedPubMedCentralGoogle Scholar
  48. Richter W, Mika D, Blanchard E et al (2013) beta1-adrenergic receptor antagonists signal via PDE4 translocation. EMBO Rep 14:276–283PubMedPubMedCentralGoogle Scholar
  49. Russwurm M, Mullershausen F, Friebe A et al (2007) Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach. Biochem J 407:69–77PubMedPubMedCentralGoogle Scholar
  50. Sato M, Hida N, Ozawa T et al (2000) Fluorescent indicators for cyclic GMP based on cyclic GMP-dependent protein kinase I alpha and green fluorescent proteins. Anal Chem 72:5918–5924PubMedGoogle Scholar
  51. Shafer OT, Kim DJ, Dunbar-Yaffe R et al (2008) Widespread receptivity to neuropeptide PDF throughout the neuronal circadian clock network of drosophila revealed by real-time cyclic AMP imaging. Neuron 58:223–237PubMedPubMedCentralGoogle Scholar
  52. Sin YY, Edwards HV, Li X et al (2011) Disruption of the cyclic AMP phosphodiesterase-4 (PDE4)-HSP20 complex attenuates the beta-agonist induced hypertrophic response in cardiac myocytes. J Mol Cell Cardiol 50:872–883Google Scholar
  53. Sprenger JU, Nikolaev VO (2013) Biophysical techniques for detection of cAMP and cGMP in living cells. Int J Mol Sci 14:8025–8046PubMedPubMedCentralGoogle Scholar
  54. Sprenger JU, Perera RK, Steinbrecher JH et al (2015) In vivo model with targeted cAMP biosensor reveals changes in receptor-microdomain communication in cardiac disease. Nat Commun 6:6965Google Scholar
  55. Stangherlin A, Gesellchen F, Zoccarato A et al (2011) cGMP signals modulate cAMP levels in a compartment-specific manner to regulate catecholamine-dependent signaling in cardiac myocytes. Circ Res 108:929–939PubMedPubMedCentralGoogle Scholar
  56. Sutherland EW, Rall TW (1958) Fractionation and characterization of a cyclic adenine ribonucleotide formed by tissue particles. J Biol Chem 232:1077–1091PubMedPubMedCentralGoogle Scholar
  57. Tao W, Rubart M, Ryan J et al (2015) A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy. Am J Physiol Cell Physiol 309:C724–C735PubMedPubMedCentralGoogle Scholar
  58. Thunemann M, Wen L, Hillenbrand M et al (2013) Transgenic mice for cGMP imaging. Circ Res 113:365–371PubMedPubMedCentralGoogle Scholar
  59. Violin JD, DiPilato LM, Yildirim N et al (2008) beta2-adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics. J Biol Chem 283:2949–2961PubMedGoogle Scholar
  60. Wachten S, Masada N, Ayling LJ et al (2010) Distinct pools of cAMP Centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells. J Cell Sci 123:95–106PubMedGoogle Scholar
  61. Warrier S, Belevych AE, Ruse M et al (2005) Beta-adrenergic- and muscarinic receptor-induced changes in cAMP activity in adult cardiac myocytes detected with FRET-based biosensor. Am J Physiol Cell Physiol 289:C455–C461Google Scholar
  62. Williams C (2004) cAMP detection methods in HTS: selecting the best from the rest. Nat Rev Drug Discov 3:125–135PubMedGoogle Scholar
  63. Zaccolo M (2004) Use of chimeric fluorescent proteins and fluorescence resonance energy transfer to monitor cellular responses. Circ Res 94:866–873PubMedGoogle Scholar
  64. Zaccolo M (2009) cAMP signal transduction in the heart: understanding spatial control for the development of novel therapeutic strategies. Br J Pharmacol 158:50–60PubMedPubMedCentralGoogle Scholar
  65. Zaccolo M, De Giorgi F, Cho CY et al (2000) A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2:25–29Google Scholar
  66. Zaccolo M, Pozzan T (2002) Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 295:1711–1715Google Scholar
  67. Zhang CL, Katoh M, Shibasaki T et al (2009) The cAMP sensor Epac2 is a direct target of antidiabetic sulfonylurea drugs. Science 325:607–610PubMedGoogle Scholar
  68. Zhang J, Campbell RE, Ting AY et al (2002) Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3:906–918PubMedGoogle Scholar
  69. Zhang J, Hupfeld CJ, Taylor SS et al (2005) Insulin disrupts beta-adrenergic signalling to protein kinase a in adipocytes. Nature 437:569–573PubMedGoogle Scholar
  70. Zhang J, Ma Y, Taylor SS et al (2001) Genetically encoded reporters of protein kinase a activity reveal impact of substrate tethering. Proc Natl Acad Sci U S A 98:14997–15002PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  1. 1.Institute of Experimental Cardiovascular ResearchUniversity Medical Center Hamburg-EppendorfHamburgGermany
  2. 2.DZHK (German Center for Cardiovascular Research) Partner Site Hamburg/Kiel/LübeckHamburgGermany

Personalised recommendations