Volume 16 Supplement 1

Abstracts from the 7th International Conference on cGMP Generators, Effectors and Therapeutic Implications

Open Access

Direct visualization of cGMP microdomains in adult cardiomyocytes

  • Hariharan Subramanian1Email author,
  • Alexander Froese2,
  • Daniela Hübscher2,
  • Konrad R Götz2 and
  • Viacheslav O Nikolaev1
BMC Pharmacology and Toxicology201516(Suppl 1):A91

https://doi.org/10.1186/2050-6511-16-S1-A91

Published: 2 September 2015

Background

cGMP is an important second messenger which regulates cardiac function and might protect the heart from hypertrophy and failure by acting in distinct subcellular microdomains. However, direct visualization of cGMP in such microdomains of adult cardiomyocytes has been challenging due to low sensitivity of the available real-time imaging techniques.

Methods

We used a highly sensitive cytosolic Förster resonance energy transfer (FRET)-based biosensor, red cGES-DE5 (affinity for cGMP ~40 nM) to generate its targeted versions and transgenic mice expressing them in adult mouse myocardium to monitor cGMP in freshly isolated adult ventricular cardiomyocytes.

Results

Cardiomyocytes were isolated from the transgenic mice expressing the cytosolic red cGES-DE5 sensor [1]. Using the previously established scanning ion conductance microscopy (SICM)/FRET method [2], local application of Atrial Natriuretic Peptide (ANP) at T-tubules and crests revealed the exclusive localization of guanylyl cyclase A (GC-A) in T-tubules. In contrast, guanylyl cyclase B (GC-B), the receptor for the C-type Natriuretic Peptide (CNP), was more evenly distributed across the membrane. Further, two differentially targeted cGMP sensors were generated. One of them was targeted to the caveolin-rich membrane microdomains via a palmitoylation and myristoylation motif (pmDE5) and another one to the sarcoplasmic reticulum by the fusion with phospholamban (DE5-PLN). FRET measurements in cardiomyocytes from these transgenic mice revealed close association of the GC-A with the caveolin-rich membrane microdomains, as well as a CNP, but not ANP-dependent regulation of cGMP levels at the sarcoplasmic reticulum.

Conclusion

FRET and SICM/FRET approaches with freshly isolated cardiomyocytes represents a powerful way of dissecting local cGMP signals in distinct cardiac microdomains. It reveals distinct cGMP microdomains associated with various GCs.

Authors’ Affiliations

(1)
Institute for Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf (UKE)
(2)
Heart Research Center Göttingen, University of Göttingen Medical Center (UMG)

References

  1. Götz KR, Sprenger JU, Perera RK, Steinbrecher JH, Lehnart SE, Kuhn M, et al: Transgenic mice for real-time visualization of cGMP in intact adult cardiomyocytes. Circ Res. 2014, 114 (8): 1235-1245. 10.1161/CIRCRESAHA.114.302437.View ArticlePubMedGoogle Scholar
  2. Nikolaev VO, Moshkov A, Lyon AR, Miragoli M, Novak P, Paur H, et al: Beta2-adrenergic receptor redistribution in heart failure changes cAMP compartmentation. Science. 2010, 327 (5973): 1653-1657. 10.1126/science.1185988.View ArticlePubMedGoogle Scholar

Copyright

© Subramanian et al. 2015

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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