Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes

Journal article

Aims Many of the complex effects of cAMP on cardiomyocytes can only be explained in terms of microdomain signaling. The spatial range of a cAMP signal is believed to be controlled by cAMP degradation, buffering and diffusivity (DcAMP). Whilst the importance of phosphodiesterases (degradative enzymes) in sculpting cAMP microdomains is well-established in cardiomyocytes, the extent to which cAMP buffering and diffusion contribute to the generation of cAMP microdomains is poorly understood. Earlier studies have suggested fast diffusion, which argues against sharp microdomain signaling. Methods and Results cAMP dynamics in adult rat ventricular myocytes were imaged using a fourthgeneration genetically-encoded sensor. The [cAMP] response to the addition and removal of isoproterenol (-adrenoceptor agonist) quantified the rates of cAMP synthesis and degradation. In paired experiments, microfluidics delivered the agonist to one half of the myocyte to generate an intracellular [cAMP] gradient, a read-out of DcAMP. After accounting for phosphodiesterase activity, DcAMP was 34 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 analog 6-Bnz-cAMP did not accelerate DcAMP, arguing against a significant role of buffering in restricting cAMP mobility. Intracellular DcAMP was no different from the diffusivity of fluorescent markers of comparable molecular size. Diffusivity was, however, faster in neonatal myocytes. Conclusions High tortuosity due to physical barriers inside adult cardiomyocytes restricts cAMP diffusion to levels that are compatible with microdomain signaling. Tortuosity can vary between different cells, and is an important factor in shaping second messenger microdomains.

Authors: Swietach, P; Richards, M; Vaughan-Jones, R; Ford, K; Lefkimmiatis, K

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Oxford University Press
• Journal: Cardiovascular Research
• Volume: 110
• Issue: 3
• Pages: 395-407
• Publication date: 2016-04-01
• Acceptance date: 2016-04-11
• DOI: 10.1093/cvr/cvw080
• EISSN: 1755-3245
• ISSN: 0008-6363
• Keywords: Microfluidics; cAMP microdomains; mathematical modelling; diffusion-reaction; FRET sensor