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Cardiovasc Res. 2016 Jun 1;110(3):395-407. doi: 10.1093/cvr/cvw080. Epub 2016 Apr 18.

Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes.

Author information

1
Burdon Sanderson Cardiac Science Centre, Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK.
2
Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, Netherlands.
3
Burdon Sanderson Cardiac Science Centre, Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK BHF Centre of Research Excellence, Oxford.
4
Burdon Sanderson Cardiac Science Centre, Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK pawel.swietach@dpag.ox.ac.uk.

Abstract

AIMS:

3',5'-Cyclic adenosine monophosphate (cAMP) signals in the heart are often confined to concentration microdomains shaped by cAMP diffusion and enzymatic degradation. While the importance of phosphodiesterases (degradative enzymes) in sculpting cAMP microdomains is well established in cardiomyocytes, less is known about cAMP diffusivity (DcAMP) and factors affecting it. Many earlier studies have reported fast diffusivity, which argues against sharply defined microdomains.

METHODS AND RESULTS:

[cAMP] dynamics in the cytoplasm of adult rat ventricular myocytes were imaged using a fourth generation genetically encoded FRET-based sensor. The [cAMP]-response to the addition and removal of isoproterenol (β-adrenoceptor agonist) quantified the rates of cAMP synthesis and degradation. To obtain a read out of DcAMP, a stable [cAMP] gradient was generated using a microfluidic device which delivered agonist to one half of the myocyte only. After accounting for phosphodiesterase activity, DcAMP was calculated to be 32 µm(2)/s; an order of magnitude lower than in water. Diffusivity was independent of the amount of cAMP produced. Saturating cAMP-binding sites with the analogue 6-Bnz-cAMP did not accelerate DcAMP, arguing against a role of buffering in restricting cAMP mobility. cAMP diffused at a comparable rate to chemically unrelated but similar sized molecules, arguing for a common physical cause of restricted diffusivity. Lower mitochondrial density and order in neonatal cardiac myocytes allowed for faster diffusion, demonstrating the importance of mitochondria as physical barriers to cAMP mobility.

CONCLUSION:

In adult cardiac myocytes, tortuosity due to physical barriers, notably mitochondria, restricts cAMP diffusion to levels that are more compatible with microdomain signalling.

KEYWORDS:

Diffusion reaction; FRET sensor; Mathematical modelling; Microfluidics; cAMP microdomains

PMID:
27089919
PMCID:
PMC4872880
DOI:
10.1093/cvr/cvw080
[Indexed for MEDLINE]
Free PMC Article

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