Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes.

Mark Richards,Oliver Lomas,Konstantinos Lefkimmiatis,Richard D Vaughan-Jones,Kees Jalink,Kerrie L Ford,Pawel Swietach

doi:10.1093/cvr/cvw080

Abstract

Aims3′,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 µm2/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.ConclusionIn adult cardiac myocytes, tortuosity due to physical barriers, notably mitochondria, restricts cAMP diffusion to levels that are more compatible with microdomain signalling.

Highlights

Signalling triggered by 3′,5′-cyclic adenosine monophosphate plays an essential role in cardiac physiology
This study provides a measurement of diffusivity, at 378C, of endogenously produced cyclic adenosine monophosphate (cAMP) in the cytoplasm of the adult mammalian cardiac ventricular myocyte, a cell type that engages in cAMP microdomain signalling
Physical restrictions imposed by cardiac myocyte ultrastructure are sufficient to explain why DcAMP in cytoplasm is considerably lower than in pure water

Summary

Introduction

Signalling triggered by 3′,5′-cyclic adenosine monophosphate (cAMP) plays an essential role in cardiac physiology. The biological effects of cAMP depend on the amount produced and the subcellular distribution of the signal. Spatio-temporal [cAMP] dynamics are determined by production by adenylyl cyclases (ACs), diffusion, buffering, and degradation by phosphodiesterases (PDEs).[1] In principle, changes to any of these factors could alter the range and extent of target activation. The importance of diffusion in shaping cAMP signals in cardiac myocytes has only been marginally addressed because of difficulties in obtaining robust diffusivity measurements. Signalling microdomains can be assembled on a framework of strategically placed synthetic, degradative, and target proteins.[2,3] Tethering of protein kinase A (PKA), the main cAMP effector, to specific subcellular compartments is achieved by A-kinase anchoring proteins (AKAPs).[2]

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Conclusion