Multiscale approach to link red blood cell dynamics, shear viscosity, and ATP release

Abstract

RBCs are known to release ATP, which acts as a signaling molecule to cause dilation of blood vessels. A reduction in the release of ATP from RBCs has been linked to diseases such as type II diabetes and cystic fibrosis. Furthermore, reduced deformation of RBCs has been correlated with myocardial infarction and coronary heart disease. Because ATP release has been linked to cell deformation, we undertook a multiscale approach to understand the links between single RBC dynamics, ATP release, and macroscopic viscosity all at physiological shear rates. Our experimental approach included microfluidics, ATP measurements using a bioluminescent reaction, and rheology. Using microfluidics technology with high-speed imaging, we visualize the deformation and dynamics of single cells, which are known to undergo motions such as tumbling, swinging, tanktreading, and deformation. We report that shear thinning is not due to cellular deformation as previously believed, but rather it is due to the tumbling-to-tanktreading transition. In addition, our results indicate that ATP release is constant at shear stresses below a threshold (3 Pa), whereas above the threshold ATP release is increased and accompanied by large cellular deformations. Finally, performing experiments with well-known inhibitors, we show that the Pannexin 1 hemichannel is the main avenue for ATP release both above and below the threshold, whereas, the cystic fibrosis transmembrane conductance regulator only contributes to deformation-dependent ATP release above the stress threshold.

Fig. 1.

A multiscale view of the links between RBC dynamics, shear viscosity, and ATP release. RBC dynamics including tumbling, tanktreading, swinging, and stretching are descriptions of the cell’s mechanical response to flow. ATP release from RBCs through transmembrane channels is a chemical signaling mechanism, which induces vasodilation in the circulation. Shear viscosity η is a macroscale measurement of the blood’s resistance to flow and a contributor to shear stress on the vessel walls.

Fig. 2.

Relating the bulk viscosity with ATP release from RBCs. (A) Viscosity parameter η/η_o versus shear stress σ for RBCs in λ = 11.1, 3.8, and 1.6 solutions, where λ is the viscosity contrast. (B) Relative ATP release versus shear stress. Changes between regimes I and II and between II and III identified as approximately 0.4 and 3 Pa, respectively, are labeled with vertical lines. Error bars =  ± 1 SEM; N = 3–7.

Fig. 3.

Microfluidic observations of single cells in flow. (A) The stretch ratio versus shear stress for λ = 11.1 and 1.6 solutions. Images display typical cells and their corresponding shear stress level. (B) The ratio of the number of cells tumbling N_tb to the total number of cells N_cell versus the shear stress. Regimes I, II, and III are as identified in Fig. 2. Error bars =  ± 1 SEM; N = 21–65.

Fig. 4.

Analysis of tanktreading of single cells. (A, Upper) Time-lapse images showing tanktreading cell in λ = 11.1 and (A, Lower) for comparison a λ = 1.6 cell. (B, Upper) Multiple outlines of both cell perimeters scaled up and overlapped showing the cell in λ = 11.1 solution swinging or wobbling; in contrast (B, Lower), the λ = 1.6 cell holds a constant orientation. (C) The radial distance to a bead on the membrane is denoted as R. Data corresponding to the images in A, R values versus time for single cells in λ = 11.1 σ = 1.10 Pa and λ = 1.6 σ = 1.85 Pa. Solid curves are fits of sin(ωt + φ). (D) Nondimensional tanktreading frequency, , where f = ω/2π, versus shear stress. Error bars =  ± 1 SEM; N = 3–8.

Fig. 5.

Inhibition of ATP release mechanism components. Relative ATP release versus shear stress for RBC solutions in λ = 1.6, controls, 100 μM carbenoxolone thought to inhibit Px1, and 50 μM glibenclamide thought to inhibit CFTR. Regimes I, II, and III are marked with arrows. Error bars =  ± 1 SEM; N = 4–7.