Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes

Mark Richards, Oliver Lomas, Kees Jalink, Kerrie L. Ford, Richard D. Vaughan-Jones, Konstantinos Lefkimmiatis, Pawel Swietach
2016 Cardiovascular Research  
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 (D cAMP ) and factors affecting it. Many earlier studies have reported fast diffusivity, which argues against sharply defined microdomains. Methods and
more » ... esults [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 (b-adrenoceptor agonist) quantified the rates of cAMP synthesis and degradation. To obtain a read out of D cAMP , 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, D cAMP was calculated to be 32 mm 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 D cAMP , 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. ---
doi:10.1093/cvr/cvw080 pmid:27089919 pmcid:PMC4872880 fatcat:nu3tze4sbzczna7lpc6msc2ozy