title: Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes.
abstract: 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.
  
  
  
  [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.
  
  
  
  In adult cardiac myocytes, tortuosity due to physical barriers, notably mitochondria, restricts cAMP diffusion to levels that are more compatible with microdomain signalling.
authors.affiliation: Burdon Sanderson Cardiac Science Centre, Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Richards
authors.affiliation: Burdon Sanderson Cardiac Science Centre, Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK.
authors.forename: Oliver
authors.initials: O
authors.lastname: Lomas
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, Netherlands.
authors.forename: Kees
authors.initials: K
authors.lastname: Jalink
authors.affiliation: Burdon Sanderson Cardiac Science Centre, Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK.
authors.forename: Kerrie L
authors.initials: KL
authors.lastname: Ford
authors.affiliation: Burdon Sanderson Cardiac Science Centre, Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Vaughan-Jones
authors.affiliation: Burdon Sanderson Cardiac Science Centre, Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK BHF Centre of Research Excellence, Oxford.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Lefkimmiatis
authors.affiliation: Burdon Sanderson Cardiac Science Centre, Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK pawel.swietach@dpag.ox.ac.uk.
authors.forename: Pawel
authors.initials: P
authors.lastname: Swietach
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000465:Algorithms; D000818:Animals; D015374:Biosensing Techniques; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer; D045325:HCT116 Cells; D057809:HEK293 Cells; D006352:Heart Ventricles / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D008929:Mitochondria, Heart / Q000378:metabolism; D008955:Models, Cardiovascular; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D010477:Perfusion; D017207:Rats, Sprague-Dawley; D015290:Second Messenger Systems / Q000187:drug effects; D013997:Time Factors
chemical_list: D000318:Adrenergic beta-Agonists; C050086:N(6)-benzoyl-cyclic AMP; D000242:Cyclic AMP; D007545:Isoproterenol
keywords: Diffusion reaction; FRET sensor; Mathematical modelling; Microfluidics; cAMP microdomains