Evidence for modulation of heat induced changes in erythrocytes by cytochalasin D: potential role for oxygen nanobubbles in aggregation

Abstract

Thermal burn injury is associated with an increase in erythrocyte (RBC) aggregation (rouleaux) prior to fibrin clot formation; unresolved rouleaux occlude vessels and contribute to thermal burn injury progression (Clark, 2013). RBC aggregation is reversible, yet the mechanism is unknown. Current models do not include all biophysical phenomena, including the role of the cytoskeleton, and sudden oxygen release from hemoglobin. We previously proposed that thermally induced membrane deformation creates nucleation sites for excess oxygen to coalesce, i.e., form nanobubbles, which may contribute to surface tension induced aggregation. To begin to address the role of the cytoskeleton in membrane deformation through changes in the band 3‐ankyrin/adducin bridge and spectrin‐actin interactions, we have disrupted actin polymerization with cytochalasin D (cytoD). We hypothesized that disrupting the cytoskeleton would alter the apparent presence of oxygen nanobubbles on singlet RBCs. Our experimental model examines both size and greyscale appearance of RBCs with sudden changes in temperature. Temperatures of 41–49 C cause shifts in brighter greyscale and larger apparent RBC size, consistent with light diffraction through concentrated gas bubbles that dissipates with time or return to 37 C (Blasco, 2017). Washed murine RBCs (obtained through a tissue sharing program) were suspended in saline (PBS) containing 1% EtOH, 0.5 uM CytoD (in 0.1% EtOH) or control, and incubated at 37°C prior to use. RBCs settled to a monolayer within an enclosed microchannel at 37°C (heated stage). The stage temperature rapidly heated from 37°C to test temperature (37, 39, 41, 43, 45, 47, or 49°C) and we imaged at 2 Hz for 70 seconds (60X, SWI). Singlet RBCs were followed over time, measuring greyscale and apparent size of the cell (outer rim) and core (central biconcave) (ImageJ). The initial changes were seen during the temperature shift and included increased core greyscale and apparent size of 20–40% between 41–47°C, with corresponding changes in cell size. Over the next several seconds there was temperature dependent fluctuations in size and greyscale of up to 50%. Cells in EtOH were similar overall to controls. CytoD prevented changes in greyscale and in size, and suppressed further fluctuations until higher temperatures where those treated cells had formed echinocytes. The data are consistent with gas bubbles forming at the RBC membrane, causing diffraction of the light, in a temperature dependent fashion. As destabilization of the cytoskeleton prevented these changes, we conclude that the cytoskeleton contributes to membrane shape change necessary for oxygen nanobubble formation with heat.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.