Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes

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.

Red blood cell thickness is evolutionarily constrained by slow, hemoglobin-restricted diffusion in cytoplasm

“…Time delays in calcein fluorescence measured at different distances from the bleaching region provide a readout of cytoplasmic diffusivity (D calc ), measured to be 47.8 ± 5.95 μm 2 /s, i.e. 13-fold lower than in water (~600 μm 2 /s)30 (Fig. 2A).…”

Cardiac sympathetic innervation network shapes the myocardium by locally controlling cardiomyocyte size through the cellular proteolytic machinery

“…In addition, our data showed that active SNs function as point-sources of NE, which consistently activate cAMP generation from the corresponding junctional membrane portion. Previously, it was reported that free diffusion of cAMP in the cellular matrix is restricted both through degradation by phosphodiesterases (Jurevicius & Fischmeister, 1996;Perry et al, 2002;Zaccolo & Pozzan, 2002;Mongillo et al, 2004), as well as by physical barriers created by the tortuous CM ultrastructure, including the positioning of mitochondria and sarcomeres (Agarwal et al, 2016;Richards et al, 2016). The obvious implication of confined subcellular cAMP signalling is that rapid and simultaneous cAMP/PKA-dependent effects (e.g.…”

Exploring cells with targeted biosensors

“…However, mathematical models predict that even with slow diffusing cAMP (∼10-35 µm 2 s −1 ) the sole PDE action is not sufficient to generate microdomains (Yang et al, 2016). These data would suggest that the formation of cAMP microdomains is likely to be a multi-parametric phenomenon where more than one mechanism such as enrichment in selected subcellular compartments of different types of adenylyl cyclases (ACs) and effectors, physical barriers (Richards et al, 2016), and cAMP buffering (Agarwal et al, 2016) that limit cAMP diffusion, along with specific localization of PDEs and PKA co-occur to ensure the existence of distinct cAMP events within the cell. Interested readers are referred to a recent review for a more detailed discussion of this topic (Feinstein et al, 2012;Saucerman et al, 2014).…”