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Adenoviral Transduction of FRET-Based Biosensors for cAMP in Primary Adult Mouse Cardiomyocytes

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cAMP Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1294))

Abstract

Genetically encoded biosensors that make use of fluorescence resonance energy transfer (FRET) are important tools for the study of compartmentalized cyclic nucleotide signaling in living cells. With the advent of germ line and tissue-specific transgenic technologies, the adult mouse represents a useful tool for the study of cardiovascular pathophysiology. The use of FRET-based genetically encoded biosensors coupled with this animal model represents a powerful combination for the study of cAMP signaling in live primary cardiomyocytes. In this chapter, we describe the steps required during the isolation, viral transduction, and culture of cardiomyocytes from an adult mouse to obtain reliable expression of genetically encoded FRET biosensors for the study of cAMP signaling in living cells.

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References

  1. Tsien RW (1977) Cyclic AMP and contractile activity in heart. Adv Cyclic Nucleotide Res 8:363–420

    CAS  PubMed  Google Scholar 

  2. Miki N, Baraban JM, Keirns JJ, Boyce JJ, Bitensky MW (1975) Purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments. J Biol Chem 250(16):6320–6327

    CAS  PubMed  Google Scholar 

  3. Northrop G, Parks RE Jr (1964) 3′, 5′-Amp-induced hyperglycemia in intact rats and in the isolated perfused rat liver. Biochem Pharmacol 13:120–123

    Article  CAS  PubMed  Google Scholar 

  4. Zaccolo M, Pozzan T (2002) Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 295(5560):1711–1715. doi:10.1126/science.1069982

    Article  CAS  PubMed  Google Scholar 

  5. Adams SR, Harootunian AT, Buechler YJ, Taylor SS, Tsien RY (1991) Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349(6311):694–697

    Article  CAS  PubMed  Google Scholar 

  6. Zaccolo M, De Giorgi F, Cho CY, Feng L, Knapp T, Negulescu PA, Taylor SS, Tsien RY, Pozzan T (2000) A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2(1):25–29. doi:10.1038/71345

    Article  CAS  PubMed  Google Scholar 

  7. Nikolaev VO, Bunemann M, Schmitteckert E, Lohse MJ, Engelhardt S (2006) Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching beta1-adrenergic but locally confined beta2-adrenergic receptor-mediated signaling. Circ Res 99(10):1084–1091. doi:10.1161/01.RES.0000250046.69918.d5

    Article  CAS  PubMed  Google Scholar 

  8. Pavlovic D, McLatchie LM, Shattock MJ (2010) The rate of loss of T-tubules in cultured adult ventricular myocytes is species dependent. Exp Physiol 95(4):518–527. doi:10.1113/expphysiol.2009.052126

    Article  CAS  PubMed  Google Scholar 

  9. Gesellchen F, Stangherlin A, Surdo N, Terrin A, Zoccarato A, Zaccolo M (2011) Measuring spatiotemporal dynamics of cyclic AMP signaling in real-time using FRET-based biosensors. Methods Mol Biol 746:297–316. doi:10.1007/978-1-61779-126-0_16

    Article  CAS  PubMed  Google Scholar 

  10. Louch WE, Sheehan KA, Wolska BM (2011) Methods in cardiomyocyte isolation, culture, and gene transfer. J Mol Cell Cardiol 51(3):288–298. doi:10.1016/j.yjmcc.2011.06.012

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Li Z, Sharma RV, Duan D, Davisson RL (2003) Adenovirus-mediated gene transfer to adult mouse cardiomyocytes is selectively influenced by culture medium. J Gene Med 5(9):765–772. doi:10.1002/jgm.405

    Article  CAS  PubMed  Google Scholar 

  12. Tian Q, Pahlavan S, Oleinikow K, Jung J, Ruppenthal S, Scholz A, Schumann C, Kraegeloh A, Oberhofer M, Lipp P, Kaestner L (2012) Functional and morphological preservation of adult ventricular myocytes in culture by sub-micromolar cytochalasin D supplement. J Mol Cell Cardiol 52(1):113–124

    Article  CAS  PubMed  Google Scholar 

  13. Eng J, Lynch RM, Balaban RS (1989) Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes. Biophys J 55(4):621–630. doi:10.1016/s0006-3495(89)82859-0

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20(1):87–90. doi:10.1038/nbt0102-87

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This study was supported by the British Heart Foundation (PG/10/75/28537 and RG/12/3/29423) and the NSF-NIH CRCNS program (NIH R01 AA18060) to MZ and Wellcome Trust PhD Programme for Clinicians at the University of Oxford to OCL.

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Authors and Affiliations

  1. Department of Cardiovascular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DU, UK

    Oliver Lomas & Ricardo Carnicer

  2. Department of Physiology, Anatomy and Genetics, University of Oxford, Sherrington Building, Parks Road, Oxford, OX1 3PT, UK

    Oliver Lomas, Marcella Brescia, Stefania Monterisi, Nicoletta C. Surdo & Manuela Zaccolo

Authors
  1. Oliver Lomas
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  2. Marcella Brescia
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  3. Ricardo Carnicer
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  4. Stefania Monterisi
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  5. Nicoletta C. Surdo
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  6. Manuela Zaccolo
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Corresponding author

Correspondence to Manuela Zaccolo .

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Editors and Affiliations

  1. Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford, United Kingdom

    Manuela Zaccolo

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Lomas, O., Brescia, M., Carnicer, R., Monterisi, S., Surdo, N.C., Zaccolo, M. (2015). Adenoviral Transduction of FRET-Based Biosensors for cAMP in Primary Adult Mouse Cardiomyocytes. In: Zaccolo, M. (eds) cAMP Signaling. Methods in Molecular Biology, vol 1294. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2537-7_8

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  • DOI: https://doi.org/10.1007/978-1-4939-2537-7_8

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2536-0

  • Online ISBN: 978-1-4939-2537-7

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